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Stellingen
1.I ti spossibl et oaccentuat eth edifference s betweentax awit hdifferen t character state definitions in such a way, that these look much more different than the taxa actually are (see Verdcourt, Verbenaceae FTEA, 1992p . 58-59).
2. Geographical isolationo fpopulation s ofa specie s alonei sinadequat e for taxonomic distinction unless it is supported by other characters, otherwise a number of ecotypes would result in endless species lists (this thesis, page 39).
3. Species cannot be fully defined, nor can it be intuitively sensed, although subjectivity is involved indecisio nmaking ;a specie si sonl ya sgoo da sth eknowledg e and insights used in its delimitation. Certainly methodologies help, so do good sense and goodjudgemen tbase d onmeaningfu l experiences, andth emor eth ebette r (Crum, 1985, Bryologist 88: 221).
4. So long as the morphological, physiological and genetic characteristics of an organism do not impose limitations to its survival, evolution or speciation will be a luxury, unfortunately nature operates on necessities only (this thesis page 91).
5. To reap the investment and the potential of Mem oak in plantations of Mt. Kenya region, thinning to reduce stem density isurgentl y necessary.
6. The frontier of science is like a mirage, the closer you get the further it moves.
7. Enforcing good governance in developing countries by withholding aid does not work; by the time the government is brought to its knees and submits to the demand, the ordinary people would be on their bellies.
8. The decision by the Bank of England to sell of part of its gold reserves to bail out theheavil y indebted, developing countries islik ecreatin g fresh woundst o temporarily relieve the pains of the old ones.
9. Waiting for something to happen is worse than when itjus t happens.
10. Necessity isth emothe r ofal lcreatio nbu timaginatio ni sth efirst ste pt o creativity.
Stellingen behorend bij net proefschrift van J.O. Ahenda; Taxonomy and Genetic Structure of Meru Oak Populations, Vitexkeniensis Turrill and Vitexfischeri Gurke , in East Africa. Wageningen, 15 September, 1999. Taxonomy and Genetic Structure of Meru Oak Populations, Vitex keniensisTurril l and Vitexfischeri Giirke , in East Africa Promotors: Dr. Ir. L.J.G. van der Maesen Hoogleraar in de Plantentaxonomie
Dr. R.F. Hoekstra Hoogleraar in de Genetica
Co-promotor: Dr. Phanuel O. Oballa Head of Tree Breeding Division Kenya Forestry Research Institute fj!T^''-V
Taxonomy and Genetic Structure of Mem Oak Populations, Vitexkeniensis Turrill and Vitexfischeri Giirke , in East Africa
Joseph Oloo Ahenda
PROEFSCHRIFT ter verkrijging vand egraa dva ndocto r opgeza gva nd eRecto rMagnificu sva nd e Wageningen Universiteit Dr.CM .Karssen , inhe topenbaa r teverdedige n opwoensda g 15 September199 9 desmiddag st ehal f tweei nd eAula . CIP-gegevens Koninklijke Bibliotheek, Den Haag
Ahenda, J.O.
Taxonomy and Genetic Structure of Meru Oak Populations, Vitexkeniensis Turrill and Vitex fischeri Gurke, in East Africa
Thesis Wageningen University With Summaries in English and in Dutch. ISBN. 90-5808-104-4 Subject headings: Meru oak, Vitex keniensis Turrill, Vitex fischeri Gurke, taxonomy, population, genetic structure.
BJBLIOTHEEK LANDBOUWUNIVERSITETT WAGENTNGEN Contents
Summary 3
Samenvatting 5
1 Introduction 7 1.1 Taxonomy and genetic diversity in relation to conservation of tropical forest trees 7 1.2 Genetic structure and mating systems of forest tree populations 9 1.3 The economic importance and exploitation of Mem oak 10 1.4 Project background 12 1.5 Project objectives 13 1.6 Thesis organisation 14
2 The taxonomy and geographical distribution of V. keniensisan d V. fischeri . 15 2.1 Introduction 15 2.2 Conspectus of the genus Vitex in East Africa 15 2.3 Brief description of V. keniensisan d V. fischeri 18 2.4 The ecology of the natural range of V. keniensisan d V. fischeri 19 2.4.1 The mountainous forests of Mt. Kenya and Nyambene hills 19 2.4.2 The woody wet savanna at the foot of Mt. Elgon and Lake Victoria basin 21 2.5 Comparison between V. keniensisan d V.fischeri herbariu m specimens 22 2.6 Morphological and cytological characteristics of V. keniensisan d V. fischeri 27 2.6.1 Morphological variation in V. keniensisan d V.fischeri see d characteristics 27 2.6.2 Seedling leaf morphology of V. keniensisan d V. fischeri 29 2.6.3 Wood characteristics of V. keniensisan d V. fischeri 31 2.6.4 Cytological characteristics of V. keniensisan d V. fischeri 35 2.7 Conclusion 38
3 Mating systems of V. keniensisan d V. fischeri 41 3.1 Introduction 41 3.2 Floral morphology and phenology of V. keniensisan d V. fischeri .... 42 3.3 Reproductive biology of V. keniensisan d V. fischeri 46 3.3.1 Viability assessment of V. keniensisan d V.fischeri polle n grains 46 3.3.2 Evaluation of stigma receptivity of V. keniensisan d V. fischeri . . 49 3.3.3 Pollination mechanisms and self-compatibility in V.keniensis an d V. fischeri 51 3.3.4 Estimates of the mating parameters of V. keniensis and V. fischeri populations 54 3.3.5 Comparison of the germination vigour of seeds from cross- and self- pollination of V. keniensisan d V. fischeri 55 3.3.6 Reproductive compatibility between the V. keniensisan d V. fischeri populations and the vigour of the resultant seeds 58 3.4 Conclusion 59
4 The genetic structure of V. keniensisan d V. fischeri populations 61 4.1 Introduction 61 4.2 Analyses of the genetic structure of V. keniensisan d V. fischeri populations 63 4.2.1 Sampling and isozyme analysis methods 63 4.2.2 Staining procedures 66 4.2.3 Isozyme analyses on samples of germinating seeds of V. keniensis and V. fischeri 68 4.3 Electrophoretic analysis on polyploidy and allozyme polymorphism in V. keniensisan d V. fischeri 71 4.4 Electrophoretic phenotypes in V.keniensis an d V. fischeri populations . 75 4.5 Genetic diversity of V. keniensisan d V.fischeri population s 80 4.5.1 Statistical analysis on the electrophoretic data 81 4.5.2 The proportion of polymorphic loci (p) 81 4.5.3 Estimates of genetic diversity of populations of V.keniensis an d V. fischeri 82 4.5.4 Genetic distance and identity of populations of V. keniensisan d V. fischeri 87 4.6 Conclusion 89
5. General conclusions and recommendations 91
Literature 105
Acknowledgements 117
Curriculum vitae 118 Summary
Taxonomy and genetic structure ofMer uOa kpopulations , Vitex keniensis Turrill and Vitexfischeri Giirke, in East Africa
V. keniensis (fam. Verbenaceae), the Meru oak, is a popular tree species in East Africa valued for its high quality, termite- and fungus- resistant timber. It is fast growing thus agoo dchoic e for commercial forestry establishment. Itsfruit s are edible and the tree is appreciated by small-scale farmers as an agroforestry species. The species isclassifie d among thethreatene d forest trees inEas tAfrica . Thebenefit s from the species and the risks of depletion looming over its natural populations have made it a priority species for conservation and improvement programmes. However, information on its taxonomic status, mating systems and the diversity of its existing gene-pool has been poor or lacking. Taxonomically, the distinction between the disjunct populations of V. keniensiso f Mt. Kenya and Nyambene hills andV. fischeri of the Lake Victoria and Mt. Elgon regions has been obscure. Studies were therefore carried outo nth especies ' morphology, cytology, mating systemsan dgeneti c structure to characterise its taxonomic status, delimit its populations and propose appropriate conservation measures and improvement breeding strategy.
The cytological, reproductive and genetic traits showed more similarity than distinction between the species populations. There was significant variation at p < 0.05 in some of the morphological traits among thepopulation s but they didno t follow the pattern of the geographical distance or ecological gradient range in which the species occurs or the current taxonomic grouping. It is therefore recommended to consider the different Vitex populations ason etaxon . Thenam e V.fischeri ha spriorit y and therefore the well-known V. keniensis(Mer u oak) falls into synonymy.
V.fischeri ( =V. keniensis) exhibitshomogam y andhig hploid y level (2n = 96).Th e flowers are pollinated by Apis mellifera(bees ) and seeds dispersed by monkeys and hornbill birds. It ispartiall y self-compatible, producing seeds by both selfing and out crossing. However, innature , outcrossing seems topredominate , thusmaintainin g the high heterozygosity. The germination of the seeds resulting from the different modes of pollination did not vary, an indication of the absence or suppression of inbreeding depression by the polyploidy. The different Vitex populations are reproductively compatible and produced viable seeds. An indication that they have not undergone reproductive isolation.
Electrophoretic analysis on progeny array of the species displayed tetrasomic inheritance and independent segregation implying that it is an autotetraploid. For the eight enzymes coding for 12loci , the species exhibited highproportio n of polymorphic loci (P) of 100% ,5. 3mea n effective number of alleles and5. 1mea nnumbe r of alleles per locus. The total genetic diversity (HT)wa s 99% and mean genetic variation within the populations (Hs) was 96.5%. The high polymorphism and total genetic diversity could be a result of high outcrossing rates, seed dispersal and polyploidy. The genetic variation between the Vitexpopulation s (GST) was lower than within them (Hs). The average values of DST and GST (3.5%) each were equally low among the species' populations showing low inter-population variation.
The pattern of morphological and genetic variation between the populations did not match the geographical distance and ecological variation. This further supports the similarity observed in the cytological traits and inheritance pattern in the species' populations. The inter-population similarity canb eattribute d tothei r commonancestr y and lacko fdivergenc e possibly causedb y the species' longlifespan , high heterozygos ity, polyploidy and or mild selection pressure. The low GST (3.5%) implied that the bulk of the species' genetic variation (96.5%) is retained within each population, therefore sampling from any of the four populations would capture a sufficiently large genepoo l of the species for conservation. However, care has tob e taken toensur e that at least two populations each from the contrasting ecological zones are included, just in case some of unique alleles in the populations were not detectable by the method used i.e. isozymes. Being an autotetraploid with up to four alleles per locus, a minimum of a hundred dispersed mother trees at least 300 m from each other can be maintained in-situ ineac h of thepopulations . Their propagules canuse d for raisingex - situ conservation stands which can also serve as living gene-bank. Samenvatting
Taxonomiee ngenetisch eopbou wva nMer uOa kpopulaties , Vitexkeniensis Turril l en Vitexfischeri Giirke in Oost Afrika
Vitex keniensis Turrill (Fam. Verbenaceae), de Meru Oak, is een populaire boomsoort in Oost Afrika, gewaardeerd omd e goedekwaliteit , end eweerstan d tegen termietene n schimmels van hethout . Degroe i vand eboo m issnel , waardoor de soort een goede keuze is voor commerciele houtaanplant. De vruchten zijn eetbaar en de boom wordt op prijs gesteld door kleine boeren in agro-forestry. De soort wordt gerekend tot de bedreigde bomen in Oost Afrika. Het nut van de soort en de risico's van achteruitgang in de natuurlijke populaties leidden ertoe dat Vitex prioriteit krijgt voor behoud en veredelingsprogramma's. Kennis over de taxonomische plaatsing, de kruisingssystemen en de diversiteit vand e bestaande genenpool was tot nu toe beperkt of ontbrak. Taxonomisch was het onderscheid vaag tussen de disjuncte populaties van V. keniensis van Mount Kenia en de Nyambene heuvels respectievelijk V. fischeri Giirke van de streken rond het Victoriameer en Mount Elgon. Omdez e redenen werd onderzoek verricht naar de morfologie, de cytologie, het kruisingsgedrag en de genetische structuur van de populaties om de taxonomische status vast te stellen, de populaties te karakteriseren en geeigende methoden voor conservering en veredeling te ontwerpen.
Decytologische , kruisings-e ngenetisch eeigenschappe n blekenmee r overeenkomst dan verschillen tussen de populaties op te leveren. De variatie tussen morfologische eigenschappen was significant (p< 0.05) maare r wasgee nsamenhan g te onderkennen met hetpatroo n van geografische afstand of ecologische gradient, nochme t de huidige taxonomische indeling. Daaromword taangerade nd everschillend e Vitexpopulatie sal s een taxon te beschouwen. De naam V.fischeri heef t de prioriteit, zodoende vervalt de welbekende naam V.keniensis to t synonymic
V. fischeri (=V. keniensis)vertoon t homogamie en een hoog ploidieniveau (2n = 96). De bloemen worden bestoven door Apis mellifera bijen, de zaden worden verspreid door apen en neushoornvogels. De boom is ten dele zelfcompatibel, zaden ontstaan zowel na zelfbestuiving als na kruisbestuiving. Van nature lijkt kruis- bevruchting te overheersen en houdt zo de hoge mate van heterozygotie in stand. De kiemkracht vanzade nontstaa nui tbeid evorme nva nbestuivin gverschild e niet, hetgeen wijst opd e afwezigheid of onderdrukking van inteeltdepressie door depolyploidie . De verschillende Vitex populaties zijn onderling kruisbaar en leverden levenskrachtige zaden, een teken dat zij nog geen reproductieve isolatie hebben ondergaan.
De electroforetische analyse van de soort vertoonde tetrasome vererving en onafhankelijke uitsplitsing, wijzende op autotetraploidie. In de acht enzymsystemen coderende voor 12 loci, vertoonde de soort een hoog aandeel polymorfe loci (P) van 100%, gemiddeld 5,3 effectieve allelen en 5,1 allelen per locus. De totale genetische diversiteit (HT) was 99 %e n de gemiddelde genetische variaties binnen de populaties (Hs) was 96.5%. De hoge graad van polymorfie en totale genetische variatie kan het resultaat zijn vand ehog emat eva nkruisbevruchting , zaadverspreiding enpolyploi'die . De genetische variatie tussen de Vitex populaties (GST)wa s geringer dan die binnen de populaties (Hs). De gemiddelde waarde van Dst en GST (beide 3.5%) was even laag onder de populaties duidende op een geringe variatie tussen de populaties.
Hetpatroo nva nmorfologisch e engenetisch evariati etusse nd epopulatie s kwamnie t overeen met de geografische afstand ertussen en de ecologische verschillen. Deze resultaten ondersteunen de overeenkomsten in cytologische kenmerken en verervings- patroon van de populaties van de Meru Oak. De overeenkomst tussen de populaties kan worden toegeschreven aan de overeenkomstige voorouders en het gebrek aan veranderingen, mogelijk veroorzaakt door de lange levensduur van de soort, hoge heterozygotie, polyploi'die en/of lageselectiedruk . Delag eG ST(3.5% ) betekent dathe t merendeel van de genetische variatie van de soort (96.5%) in iedere populatie aanwezig is, zodat bemonstering van welke van de vier populaties dan ook een genenpool van voldoende diversiteit oplevert. Desalniettemin is het aan te raden dat tenminste twee populaties van de twee contrasterende ecologische zones worden bemonsterd, voor het geval dat unieke of zeldzame allelen in de populaties niet door de gebruikte methode, isoenzymanalyse, werden ontdekt. Als autotetraploid met tot vier allelen per locus volstaat het in-situ instandhouden van een minimum van een honderdtalve ruiteenstaand ebomen .Hu nzade nkunne nex-sit uboomgaarde noplevere n die als levende genenbank dienen. 1 Introduction
1.1 Taxonomy and genetic diversity in relation to conservation of tropical forest trees
Global concern over the conservation of biodiversity is manifested in the high political profile of the topic, and in the number of activities related to conservation strategies such as organisation of conferences and meetings, the involvement of many national and international institutions, and the increased levelo f scientific research and documentation. However, despiteal lthes e efforts, only asmal l fraction of biodiversity so far is under proper conservation, especially in the tropics. Currently, many ecosystems are threatened by mismanagement and adverse environmental as well as socio-economic changes. Tropical forests in developing countries hold many species that have not been described, unfortunately they are under increased pressure from deforestation and cultivation (Adams et al., 1980). Currently, forests are among the most threatened ecosystems inth eworl d (National Research Council, 1991; Williams, 1991). The benefits of forests and trees, both in ecological terms and as a source of forest products cannot be overemphasised (Bawa & Krugman, 1991), thus the questions currently being raised do not concern any longer the rationale for forest conservation and improvement programmes, but rather how they can be effected. While the population of the industrialised temperate nations are expected to increase by 30 %ove r the next 30years , those inth e nations of thetropic s will double (Ashton & Bawa, 1990). This fact has a profound implication to sustainable management of forest ecosystems.
Asnatura l forest resources decline, there isa nincreasin g needt oresor tt oplantatio n and on-farm forestry. The main obstacle to this is lack of knowledge on the biology of most of the tropical forest trees and their ecosystems. The majority of the highly exploited tropical forest are not under any conservation or improvement breeding programme. Currently, about 500 tree species occur in well-managed forests; even fewer (approximately 60)worldwid e areinclude d inwell-establishe d tree improvement programmes (National Research Council, 1991), andth eremainder , mosto f which are tropical species, are exploited without explicit consideration for their conservation. This trend poses a threat to the highly demanded forest tree species especially in the tropics. The amount of species the world can afford to lose is unknown, but it is certain that the tolerance to species over-exploitation and loss is limited. Thus, the need for strategies for sustainable utilisation of plant diversity and their genetic resources is urgent. This requires exploration of their natural range and definition of their taxonomic status, genetic distribution pattern and mating systems. Insufficient information on genetic structure, reproductive biology and pollination ecology have constrained the conservation of most tropical forest tree species and their inclusion in breeding programmes (National Research Council, 1991). Population genetic theory predicts that the decrease in the genetic diversity limits a species' ability to keeppac e with the changing selectionpressur e (e.g., Young et al. 1996). Plant species, especially the perennials such as trees, rely on the available genetic diversity for stability and survival under the ever-changing environments (National Research Council, 1991). This variability forms the base on which natural selection may act and has been exploited ever since the first domestication by man for breeding purposes. Genetic studies by Namkoong (1991), Epperson (1992), Hamrick et al. (1992) and Loveless (1992) have characterised parameters of plant genetic resources as dynamic and complex which emphasises the need for a sound approach to sampling for conservation and utilisation of species variation. Theunderstandin g of the species population structure and potential genetic resources is essential for their conservation planning and sustainable management (Sune tal. , 1998). Inth epas tmos t of the information was either ignored or gathered through limited inventory followed by establishment of provenance (field) trials which were both laborious and slow. Recent development of biochemical and molecular techniques to answer some of the basic questions have supplemented and hastened theproces s of information gathering, reducing the time taken inplannin g thebes t sampling strategies for specific purposes.
In Kenya more efforts are now being directed towards the vulnerable, highly demanded indigenous forest tree species with the aim of promoting their propagation on-farm and inplantations , natural regeneration and conservation (both in-situ andex- situ). V.keniensis (commonl y referred to as Meru oak) is one of the indigenous forest tree species in Kenya, rated highly for both commercial and on-farm production and, conservation. Currently, it is threatened by rapid exploitation for its quality timber. The number of individuals of the plant species remaining in the natural forests is small and is further dwindling. The species has been under selective logging for a long period of time subjecting it to genetic erosion. Thus its populations are threatened (Noad &Birnie , 1989; ICRAF, 1991). The demand for V.keniensis timbe r in Kenya outstrips the existing populations most of which are natural. Thus promotion of productive commercial plantations and on-farm planting supported by improvement breeding programmes is called for. That is expected to divert or reduce the pressure on the remaining trees in the natural forests. These improvement and commercial production efforts need to be coupled with both ex-situ and in-situ conservation strategies for the hardwood tree species.
However, the basic but essential information required for the initiation of its propagation and conservation programme such as its taxonomic status, mating system and the diversity of Meru oak genetic resource is still sparse. Taxonomy plays a critical role inspecie s management since only formally named entities canb e afforded legal protection (Avise, 1994). Moreover, taxonomic uniqueness has been promoted as a factor leading to priority conservation status. Thus, elucidation of genetic relationships among taxa may help define taxonomic entities and determine conservation priorities (Stewart et al., 1996). Despite the recent trend to place many Verbenaceae genera in Lamiaceae, for this thesis Iwil l keep the family as recognized in the Flora of Tropical East Africa, and keep the genus Vitexi n the Verbenaceae family. V.keniensis i s so closely related to V.fischeri tha t it is doubtful whether they belong to different species (Dale & Greenway, 1961). Frequent confusion is encountered in literature dealing with the "two species". Their leaves and flowers are hardly distinguishable. Up to now, the distinction between the two was obscure and their separation has been based mainly on their habitats and geographical location: V. keniensis hasbee n considered amontan e moist evergreen forest species andV. fischeri a wet savanna woodland species occasionally growing in forest margins. Figure 1.1 & 1.2 shows the natural habitats of the Vitex populations. The species' populations in Mt. Elgon and Lake Victoria regions have been referred to as V. fischeri by some authors but others have synonymised itwit hV. keniensis (Appendix 2.1). Thisha s led toconfusio n inth e taxonomy of species thus causing problems indeterminin g its exact population size, distribution and consequently its gene-pool for sustainable manag ement. The "two species" have a wide range of occurrence in East Africa, mostly concentrated in Mt. Kenya and Nyambene hills of central Kenya, Mt. Elgon and Lake Victoria regions in Kenya, Uganda and Tanzania. The study was therefore designed to clarify the taxonomic status of V. keniensis and V.fischeri, an d their natural range of distribution, mating systems and the genetic structure of their populations. The information isexpecte d tocontribut e to sustainable management of thespecie s through sound conservation, improvement breeding and propagation programmes.
1.2 Genetic structure and mating systems of forest tree populations
The amount of genetic variation within a species and its distribution within and between populations may provide clues to factors that govern the maintenance of variation and geneflow (Ayres & Ryan, 1999). These factors are useful in delimiting populations, identifying potential selection domain and measuring genetic diversity. The knowledge of thegeneti c diversity and mating systems of a tree species gene pool is crucial to its conservation, improvement and management of its plantations. However, like in most tropical forest tree species, information on the biological characteristics V.keniensis i s sparse. The mating system of aplan t species has adirec t influence on its gene flow and the genetic structure of its populations (Maki et al., 1999). The two are important factors to plant breeders and conservationists. The variation in the genetic structure and mating systems in different species and even populations are also taxonomically valuable and may be used in taxonomic characterisation. In recent years, isozyme electrophoresis techniques have been used widely to evaluate taxonomic conclusions of different authors, genetic relationships of tree populations and in the understanding of their mating systems (e.g. Chase et al. 1995; Ford et al. 1998;Williamso n et al., 1999; Maki et al. 1999). Such studies have been generally employed inrespons e totropica l deforestation and itsconsequence s i.e. loss ofbiodiversit y and thepotentia l losso f geneticdiversit y withinspecie s (Figure1. 3 & 1.4).
The specific traits such as life form, reproductive biology, seed dispersal and geographical range of distribution can affect the level and partitioning of genetic 5m ^^^^^^^^HHHHK!V'<
Figure 1.1. The natural habitat of V. keniensisi n Kenya. diversity of atre epopulatio n (Massey &Hamrick , 1998;Su ne t al., 1998).Thus , they are important in the designing of sampling and management strategy for conservation programmes. Outcrossing plant speciesten d tomaintai nhig htota lgeneti cdiversit y but with a higher proportion of it maintained within individual populations (Hamrick & Godt, 1989).Burle y etal . (1986)conclude dtha tmainl y self-incompatible, out-crossing species exhibit acombinatio n of clinal variation, in response tobroa d climatic factors, anddiscontinuou s variation, whendistribute d overdisjunc t environmental factors such as soil and relief. Thus data on the genetic diversity and mating systems of Meru oak populations would betherefor e be important inpredictin g geneflow anddesignin g both in-situan d ex-situconservatio n strategies. Gene bank managers and researchers also need such information to successfully collect, conserve and study such a threatened plant species. The results of the species' mating systems and genetic structure should also cast some light on the level taxonomic similarity and distinction between V. keniensisan d V.fischeri.
1.3 The economic importance and exploitation of Meru oak
V.keniensis i s one of the most popular and exploited indigenous forest tree species inEas t Africa. Itproduce shig hquality , termite-an dfungus-resistan t timber. Itstimbe r is widely used in furniture, cabinets andjoiner y in East Africa (Benghou, 1971). The current demand for its timber outstrips the production level of the existing natural populations and commercial plantations of the tree species mainly in the Mt. Kenya region (Kigomo, 1985). Besides itshig h quality timber, the tree species is appreciated
10 Figure 1.2. The natural habitat of V.fischeri i n Tanzania. for its edible fruits and medicine for stomach ailments, the flowers arepopula r asbee - forage for quality honey production. These uses are still at subsistence level but they have high economic potential especially to the rural areas. The timber is rated highly because of its light weight and light brown colour with attractive wavy grain.
Meru oak is recommended for plantation establishment especially in areas prone to elephant damage. This isbecaus e its leaves and bark are unpalatable to elephants thus making foraging damage less likely. As a high value multipurpose tree species, it is appreciated by small scale farmers for its compatibility with agricultural crops. It provides the associated crops such as maize, coffee, yams, cassava, sweet potatoes, beans and bananas with shade during the hot, dry season and mulch from its shed leaves in cold, rainy periods. It is able to withstand lopping and pollarding, thus providing the farmers with fuelwood while allowing the main stem to reach merchant able size. The tree species, therefore has a substantial potential for agroforestry. Surveys on the Vitexpopulation s indicated that their number is dwindling and their natural range of occurrence shrinking due to exploitation and destruction of their natural habitats (Figures 1.1 - 1.4). The causes of the exploitation vary from one region to another. In Mt. Elgon and Lake Victoria, Vitex populations are under threat from clearing the habitats for human settlement and plantation agriculture, mainly sugar cane and tobacco growing. In these regions the species is also exploited for charcoal and firewood for curing tobacco and domestic cooking. Its timber is also popular for making tool handles and oxen yokes. InMt . Kenya and Nyambeneregion s the species is exploited for its valuable timber and habitat destruction through human settlement, agricultural activities and forest fires which are also common.
11 Figure 1.3. Conversion of the natural habitat of V. keniensisi n Kenya, to plantation forestry.
1.4 Project background
The variety of uses of V. keniensis(Mer u oak) qualifies it as a multipurpose tree species. Its wide range of distribution over different ecological zones may be an asset in its potential for large scale improvement and propagation. Any achievement in increasing the productivity of the tree species will raise the living standards of the people whodepen d directly on itsproduct s and other benefits. Inadditio n tothat , non- wood products such as fruits and medicine, have potential commercial value. Good progress in commercial production would also alleviate the current exploitation pressure onth eremaining , dwindlingnatura lpopulation s ofth etre e species in different parts of East Africa.
Most tropical forest tree species, including V. keniensis have not been studied beyond taxonomic level; further, thetaxonom y itself often requiresa thoroug h revision asdifferen t forms areencountere d amongdistinc t speciespopulations . Currently, there are difficulties in distinguishing V.keniensis fro m V.fischeri. A n integrated approach was therefore be required to compare the possible pattern of phenotypic variation in relation to genetic diversity andpossibl y environmental conditions. Genetic evaluation and characterisation, and elucidation of mating systems of the species will form the basis for its conservation, improvement breeding, propagation and utilisation region ally. The information isals o of more general scientific interest because of its probable contribution to the understanding of the biology of other tropical plant species.
12 Figure 1.4. Conversion of the natural habitat ofV. fischeri i n Tanzania, to other land uses.
1.5 Project objectives
Themai n objectives of theresearc h project weret odetermin e thetaxonomi c status, genetic variation and mating systems of Meru oak populations for the purpose of generating information relevant to its sustainable management through scientifically based conservation, improvementbreedin g andpropagatio n strategies. Thisi sexpecte d to enhance the preservation of the species' existing natural populations, and its productivity in commercial plantations and small scale farms ensuring the timber demand is met from the planted trees.
Specific objectives 1. to elucidate the taxonomic status of V. keniensis (Meru oak) and the closely related V. fischeri for identification and establishment of its population size and natural distribution range. 2. to understand the mating systems of Meru oak. 3. to determine the genetic variation within and between the populations of Meru oak across its natural range. 4. to recommend possible sustainable management strategies for Meru oak and the other related forest tree specieso nth einformatio n gained inthi sresearc h project.
13 1.6 Thesis organisation
The thesis is presented in 5chapters . Chapter 1cover s the background information on the subject. Chapter 2present s the taxonomic distinction and similarity between V. keniensis and V. fischeri based on the study of the existing literature, herbarium specimens, morphological and cytological traits. Chapter 3 covers the reproductive biology and the mating systems of the species in the different geographical locations. Thegeneti c structure of thespecies ' populations ispresente d inChapte r 4. Thegenera l conclusion, recommendations for sustainable management of the species and suggestions for further research are given in Chapter 5.
14 2 The taxonomy and geographical distribution of V. keniensis and V. fischeri
2.1 Introduction
The genus Vitexbelong s to the family Verbenaceae and is composed of over 130 species. It has a wide distribution in Africa, Asia, Australia, New Zealand, South America and the Caribbean. Africa has about 60 species of Vitexwit h most of them occurring in the sub-saharan regions of West, East and Southern Africa including the Islands of the Seychelles and Madagascar. A few of them are found in the mediterra nean regions of northern Africa. In Asia the genus is widespread in the tropical and sub-tropical regions of south west Asia and the Middle East. There are about 35 species of thegenu s inAsi awhil e Australia andNe w Zealand havethre e Vitex species. South America and Caribbean Islands have over 40 species of Vitexspreadin g from Panama, Puerto Rico, Jamaica to Venezuela.
Some of the species in thegenu s sucha s V.agnus-castus , V.ferruginea Schum. , V. negundoan d V. altissima have a wide distribution across the world. There are about twenty different Vitex species occurring in East Africa. The uses of the species in the genus vary from one continent to another but they include timber, medicine, food (fruits and production of honey), mulch, shade and ornamental purposes.
2.2 Conspectus of the genus Vitex in East Africa
Verdcourt recently (1992) revised the genus Vitexfo r the Flora of Tropical East Africa. Not all species are sufficiently known, most do not play a major role in the timber trade. For akey , detailed descriptions, synonyms andspecimen s examined may be referred to his 1992 revision in FTEA {Verbenaceae). Shorter keys, maps and descriptions are given by Beentje (1994). The species are given in alphabetical order. Figure 2.1 shows the natural of distribution of the species in the genus Vitexi n East Africa.
1. Vitexagnus-castus L . Deciduousornamenta l shrubo rspreadin g tree, oncecollecte d incompletely north of Dar-es-Salaam.
2. V. amaniensis G. Piep. Tree 12 to 20 m tall, Lushoto district, Morogoro district in Tanzania, forest, forest edges and relict, syn. V.lokundjensis sensu Moldenke, pro parte, non Pieper.
3. V. buchananii Giirke. Herb, shrub or branched tree 1.2 to 15 m tall, Lushoto district in Tanzania, also Malawi, Zambia, Mozambique.
15 o IOO ice :oo
Figure 2.1. Natural distribution of the genus Vitexi n East Africa.
16 4. V.doniana Sweet . Tree 3.5 to 15 mtall , deciduous, branched, inwoode d grassland or forest edge, commonest savanna tree and one of the most useful member of the family, in Teso Bunyoro districts of Uganda; Mt. Elgon, South Nyanza, Kuria and Kwale districts of Kenya; Bukoba, Kondoa, Morogoro districts and Zanzibar in Tanzania. Also throughout western and southern tropical Africa. Its wood isuse d for making furniture, dhow ribs, canoes and house-building.
5. V. ferruginea Schum. (include V. amboniensis Giirke, V. carvalhi Giirke, V. tangensisGiirke) . Shrub or tree 1.5 - 6 m, forest (margin), secondary bushland or thicket, evergreencoasta lbushland .Verdcour tdistinguishe ssubspecies ferruginea from Uganda, Tanzania and Kenya, and subspecies amboniensis(Giirke ) Verdcourt from Tanzania, also in Somalia, Mozambique, Zambia, Zimbabwe and South Africa.
6. V.fischeri Giirke. Tree 3 - 15m , wooded grassland less often in forest margins, itstimbe r isuse d for making furniture and oxenyokes , and its fruits are edible, occurs in Teso, Busoga and Mengo districts Uganda; Trans-Nzoia, Kakamega and north Kavirondo districts of Kenya; Mwanza, Shinyanga and Kigoma districts of Tanzania, also inZaire , Sudan, Zambia andAngol a Savannawoode d grassland, syn.V. keniensis sensu T.T.C.L.: 643 (1949), non Turrill.
7. V. keniensis Turrill (syn. V. balbiChiov.) . Tree 12 to 35 m tall, moist evergreen forest sometimes riverine in SouthNyeri , Meru, Nyambene hills, Taita hills inKenya , cultivated in Uganda and Tanzania, produces superior termite fungi resistant timber and fruits are edible, often synonymised with V.fischeri, se e conclusion inchapte r 2.
8. V. madiensis Oliv. Shrub or small tree, 1.2 to 7.5 m tall, with rough bark, or pyrophytic herb to small shrub 0.3-1.5 m tall, forming patches 1 m wide from a massivesubterranea n woody rootstock. Verdcourtdistinguishe s subsp. madiensisfro m Uganda and many tropical African countries, subsp. milanjiensis(Britten ) F. White var. milanjiensis from Tanzania, and var. epidictyodes (Pieper) Verde, a pyrophytic shrubby herb from Tanzania, and Congo to Cabinda.
9. V. mombassae Vatke. Shrub or tree 1.8 to 6 m in Kwale Kenya, Tanga Mbulu districts of Tanzania, also in Democratic Republic of Congo, Burundi, Mozambique, Malawi, Zambia, Zimbabwe, Angola and South Africa.
10. V. mossambicensisGiirke . Shrub or small tree 4 to 18m in coastal areas, Kilwa and Lindi districts of Tanzania, also in Mozambique.
11. V.payos (Lour.) Merr. var. payos, var. glabrescens (Pieper) Mold. 2 to 9 m in Machakos, Kwaledistrict s ofKenya , Handeni, Morogoro, Kilwadistrict s of Tanzania, also in Mozambique, Malawi, Zimbabwe.
12. V. schliebenii Moldenke. Shrub of 2.4 to 3 m, in Tana River and Lamu districts of Kenya, Kilwa, Masasian dLind idistrict so fTanzania , alsoi nMalawi ,Mozambique .
17 13. V.stricken Vatke &Hildebrandt . Erect or scrambling shrub or small tree, 1.5 to 4.5 mtall , Kiambu, Teitaan dKilif i districts inKenya ; Mwanza, Tanga and Bagamayo districts in Tanzania.
14. V. trifolia L. Shrub or small tree up to 6.5 m tall, sometimes creeping, Kwale district in Kenya; Uzaramo district in Tanzania, also other parts of Africa, Asia and Australia.
15. V. ugogoensisVerde . Shrub or tree 1.5 to 5 m tall, deciduous thicket, bushland in Kondoa and Dodoma districts of Tanzania.
16. V. zanzibarensis Vatke. Shrub or tree or liana in coastal thicket or dry forest, especially at the edges. Kwale district in Kenya, Uzaramo and Kilwa districts in Tanzania, 0 to 10 and also 360 to 600 m. Probably also in Mozambique.
17. V. sp. A. Related to V. buchananiishru b with older stems hollow, Morogoro district, Tanzania.
18. V. sp. B. 18m , Uzaramo district, Tanzania, probably close toV. mossambicensis.
2.3 Brief description of V. keniensis and V. fischeri
V. keniensis and V.fischeri bot h belong to the Verbenaceae family. The common names of V. keniensis are Meru oak (English), Muhuru (Kikuyu) and Moru (Meru). Meanwhile, V. fischeri is commonly called Mohutu (Nandi), Jwelo (Luo), Mufutum- bwe, Muhutu (Luhya), Omuhuruhuru (Taita), Mhunda (Zanaki) and Kabrampako (Kitongwe). The local names of the species tend to go by the language groups and regions. The distinction between the Vitex populations has been unclear (Dale & Greenway, 1961), therefore the description presented below applies to both of them.
V.keniensis an d V.fischeri; a small to abi g tree reaching a maximum height of 30 mtal l and 2.3 mdiamete r depending on the environment, deciduous with dark brown fissured bark, sapwood light brown and heartwood dark brown, coarse-textured with well-marked growth zones, often with wavy grain figure. The wood seasons, works and nails well. Young branches and petioles are covered with orange brown velvety hairs; palmate leaves are 5-foliolated and scented; the 5 leaflets are oblong, obovate- elliptic or elliptic measuring 5 to 19 cm long, 3 to 10 cm wide, acuminate at apex, cuneate to rounded at the base, glabrous above but velvety woolly tomentose beneath with glands, venation evident, up to 15pair s of lateral nerves; petioles 6.5 to 16.5 cm long; petiolules 1 to 8c m long. Cymes axillary with many flowers up to 15c m wide, peduncle 6 to 14c m long; pedicel up to 1m m long but of the central flower of cyme up to 12m m long; calyx truncate or toothed up to 1 mm long; corolla white with blue or purple or mauve upper lip, yellowish white tube up to 3 mm long, the lip 3.5 mm long and the lower lobe 2.5 mm long and wide; fruit a pulpy drupe, black when ripe, oblong-globose up to 1.3 cm long, 1 cm wide, edible; seeds are nuts with calycine cup up to 1c m wide and 2 cm deep; containing 1t o 4 embryos. Figure 2.2 shows the leaves, flowers and fruits of V. keniensis.Furthe r descriptions and comparisons made by different authors between the "two species" are presented in appendix2.1 .
2.4 The ecology of the natural range of V. keniensisan d V. fischeri
The observations made during the field work indicated that the "two species" (V. keniensisan d V. fischeri) occur in different habitats (Figure 1.1 & 1.2), ranging from the wet, cool, high altitudes of Mt. Kenya and Nyambene hills to low-lying drier, warmer areas around thebasi n of Lake Victoria and lower slopes of Mt. Elgon. These populations are disjunct. The populations in the evergreen montane forests of Mt. Kenya and Nyambene hills arecommonl y referred toa s V.keniensis whilethos e inth e low lying wooded savanna of the foot of Mt. Elgon and Lake Victoria belt are considered to be V.fischeri (Verdcourt, 1992; Beentje, 1994). These zones have markedly different ecological conditions. The ecological zones and the habitats occupied by the different the four populations of Vitex are described below.
2.4.1 The mountainous forests of Mt. Kenya and Nyambene hills
The central highlands of Kenya of Mt. Kenya and Nyambene hills are mainly of tertiary and recent volcanic origin, dominantly represented by kenyte which is the parent material of the soils inthi s region. The soils arebrow n loam (ferrisols), mostly up to 175 cm deep. The streams are perennial and the valley floors have dark brown friable clayfrequentl y mottledb yseasona lflooding . Thegenera lphysiograph y includes mountain foot ridges, upper uplands, upper scarps, plateaus and scarps, all belonging to volcanic landscape (Morgan, 1973). In this region the populations of Meru oak occur at an altitude range of 1300 to 2000 m.
In this region, the climate tends tovar y withth e altitude; the rainfall increases with altitude while the temperature drops. The climatic conditions of the areas whereVitex occurs are shown in appendix 2.2. The rainfall here is concentrated in two seasons; the first isterme d as "warm" due to relatively hightemperature s and lasts from March toMay . The second rains fall between October and December and that iswhe nmatur e fruits of Vitexar e shed from the crown as are the dead leaves. The dry season also shows seasonality and has an influence on the plant phenology. A short cool, dry season occurs from January to February, when flowering and pollination take place and a long warm, dry season lasts from June to September when the fruits mature (the species' mating system is presented in chapter 3).
The Vitex of Mt. Kenya and Nyambene hills occur in association with Prunus africana (Hook.f.) Kalkm. (Pygeum africanum Hook.f.), Ocotea usambarensis Engl., Celtis africana Burm.f., Podocarpusfalcatus Mirb. (P. graciolor Pilger), Olea
19 Figure 2.2. Leaves, flowers and fruits of V. keniensis(Dal e & Greenway, 1961). a) flowering branchlet b) fruits c) flowers d) longitudinal section of flower (x 0.5). capensis L.,Aningeria adolfi-friederici (Engl.) Robyns &Gilb. ,Fagaropsis angolense (Engl.) Dale, Polyscias kikuyensis Summerh., Antiaris toxicaria (Pers.) Lesch., Cassipoureamalosana (Bak. )Alston ,Tabernaemontanapachysiphon Stap f( =T. holstii K. Schum.), Strombosia scheffleri Engl., Premna maximaT.C.E . Fries andXymalos monospora (Harv.) Warb. Theuppe r limit of Vitex isdetermine d byth e frost-line. The zone of Vitex in Mt. Kenya and Nyambene hills may be described as montane rain
20 forest. The level above the altitudinal range of Vitex is dominated byPodocarpus latifolia (Thunb.) Mirb. (P. milanjianus Rendle), Podocarpus falcatus Mirb. (P. graciliorPilger) ,Juniperusprocera Endl., Hageniaabyssinica (Bruce) J.F. Gmel.an d Arundinaria alpinaK . Schum.
The forest canopy in the Vitex zone is complex and multi-layered. The trees of the upper strata are 30 to 40 m tall and is dominated by Ocotea usambarensis.Vitex and the associated species form the second layer between 20 and 35 m. The shrub layer is 2 to 6 m tall, dominated by Galiniera saxifraga (Hochst.) Bridson. The forest floor is normally open with little undergrowth except in "gaps" arising from dead or felled trees. The forest floor vegetation consist of broad-leaved forest grasses, several ferns and regenerating tree species. Most of regeneration of Vitex occur in the gaps that is where thecanop y isopened , most likely becauseo f the light andwarmt h insuc hspots . The main disturbances the species is subjected to in the montane forests of Mt. Kenya and Nyambene hills are tree felling, falling dead trees and sometime forest fire. The natural regeneration of Vitex isgenerall y low inth e closed forest. This isprobabl y due to shady and cold conditions on the forest floor and seed predators. In natural forests Meru oak seems to generate in canopy gaps (Konuche, 1994), because the species is a light demander (Kenya Forest Department, 1969), thus needing crown opening to regenerate. Most of Vitexfruit s shed from the crown are eaten by the forest floor animals attracted by the sweet pulp. The depulping of the fruits by monkeys and hornbills tend to improve the germination of the resultant seeds and make the seeds less attractive to forest fungi, rodents and insect pests. Some of the animals especially insect pest burrow the seeds, damaging the embryos thus interfering with their germination reducing natural regeneration rate of the species in the nature. And since the forest floor remains shady, moist and cold most of the year, the regeneration rate remains low and most seeds do rot.
Mature trees of Vitexar e getting fewer inthes e regionsbecaus e of over-exploitation. In the Vitex zone lianas grow to aheigh t of about 20 m. In the valleys tree ferns such as Cyathea manniana Hook, andwil dbananas ,Ensete ventricosum (Welw.) Cheesman are common. However, the vegetation bordering the streams was somewhat different and had a lot of ferns but almost no trees. The vegetation there is under constant disturbance by wild animals such as elephants which frequent the areas for water and low plants, thus enabling the growth of the secondary and ruderal species.
2.4.2 The woody wet savanna at the foot of Mt. Elgon and Lake Victoria basin
The regions around Lake Victoria and Mt. Elgon where Vitex occurs are predominated by pre-cambrian granite with a variety of volcanic tuff and lava. The soils are mainly yellow-red ferralitic, fersialitic and ferruginous tropical soils. The physiography is undulating with a number of drainage channels flowing towards Lake Victoria (Morgan, 1973). The vegetation here may be described as wet-wooded savanna which isa grasslan d with scattered or grouped trees and shrubs providing less
21 crown cover. The trees are more branched with crowns that do not form a complex deep canopy. A number of tree and shrub species here including Vitex,ar e deciduous and shed their leaves during some parts of the year. The plant species occurring alongside Vitexi n these areas are Combretum molleG . Don, Albiziagummifera (J.F. Gmel.) C.A. Sm., Diospyros abyssinica (Hiern) F. White, Commiphora africana (A. Rich.) Engl. [C. pilosa (Engl.) Engl.], Milicia excelsa (Welw.) C.C. Berg., Teclea nobilis Del., Trichiliaroka Chiov. , Terminalia browniiFresen.,Vzte t donianaSwee t and Piliostigma thonningii (Schum.) Milne-Redh. In some riverine areas with less human activities, Olea europeaL . ssp. africana (Mill.) P. Green, Prunus africana (Hook.f.) Kalkm. and Warburgia ugandensis Sprague alsooccu r along side Vitex. The trees grow up to 20 m tall, with crowns just touching to form an open canopy. The Vitex in this region is capable of growing even taller if itwer e not for human activities such aspollardin g andpruning . Thestunte d growth habit reported on the Vitex inLak e Victoria and Mt. Elgon region is mostly a result of human disturbance.
The forest floor in the woody wet savanna is mostly open and is dominated by grasses andherb s such asAndropogon, Brachiaria,Chloris, Eragrostis,Themeda and Setariaspecies . The altitude of this region reaches a maximum of 1500 m above sea level. The region is highly populated leading to rapid deforestation to pave way for settlement and farming activities, thus the wooded grassland. Many of the islands in Lake Victoria such as Lolui and Sese Islands also have forests with Vitex populations. The vegetation in Lake Victoria and Mt. Elgon areas is also subjected to frequent deliberate fires to promote growth of grass for livestock. Most of the areas in these regions are being cleared for growing of cash crops like sugar-cane, tobacco and cotton, and food crops such as maize, beans, sorghum, millet, sweet potatoes and cassava. The land cleared of natural vegetation is covered to a large extent by crops and post-cultivation vegetation. Appendix 2.2 shows theclimati c conditions prevailing in Mt. Kenya, Nyambene hills, Lake Victoria and Mt. Elgon, the natural range of the Vitextre e populations.
2.5 Comparison between V. keniensis and V.fischeri herbarium specimens
The label information on sheets of V.fischeri an d V.keniensis a t e.g. EA revealed difficulties in the distinction between the two species. Studies on the EA sheets indicated that V.fischeri occurre d widely in Tanzania, Uganda and Kenya. However, labels on some sheets indicate that V.keniensis occurre d alongside V. fischeri in the same areas mentioned above. It was therefore difficult to distinguish the species by origin when using herbarium specimens.
The V.keniensis sheet s at EA indicated that the collections came from Kakamega, Chogoria in Mt. Kenya, Kitale, Gogoni Forest Reserve (Kwale), Port Victoria (at the shore of Lake Victoria), Nyambene hills, Ngaongao Forest in Taita Taveta and Meru on Mt. Kenya. But also from Shinyanga in Tanzania and Serere, Teso district in Uganda. In many cases, V. keniensis and V. fischeri were used interchangeably by
22 collectors and some authors; an indication of difficulties faced by the collectors to distinguish theplan t species onthei r morphology. It isclearl y possible that thetw o are either closely related or they are the same taxon.
Thema p infigur e 2.3 showsth enatura l rangeo fdistributio n of theV. keniensis and those identified by some authors as V. fischeri populations identified in the three countries of East Africa. Appendix 2.2 shows data on the climatic conditions of the areas where V. keniensisan d V. fischeri populations occur. They were areas around Lake Victoria basin of Kenya, Uganda and Tanzania, at the foot of Mt Elgon, central Kenyahighland s around Mt. Kenyaan d Nyambenehills . InTanzani a mosto f the Vitex is concentrated around Lake Victoria (Musoma, Bukoba and Mwanza). According to herbarium labels they also occur inShinyanga , Morogoro, Kigomaan d Iringa regions. No Vitex trees were collected from the north-eastern mountains of Tanzania (Usambaras Mountains and Mt. Kilimanjaro) contrary to earlier reports. The V. keniensisspecimen s collected from Arusha and Mgamba (Lushoto) in Tanzania were not from nature but from plantations raised from seeds collected from Mt. Kenya's Vitexaccordin g to records and the forester in Lushoto (pers. communication).
The species was also not found in Taita and Shimba hills near the Kenyan Coast, contrary to some previous reports on exploration of Ngaongao and Ngongoni forests. The Vitex populations in these areas might have been depleted by over-exploitation or habitat destruction. The Kenyan coastal forests also did not have any V. keniensiso r V.fischeri. However, there are scattered populations of Vitex doniana Sweet, V. mombassae Vatke, V.stricken Vatke and Hildebr., V. ferruginea Schum., V. trifolia var. bicolor (Wild.) Moldenke and V.payos (Lour.) Merr.
According to Dale & Greenway (1961), V. keniensis naturally occurs in the Mt. Kenya region andcollection s away from that regionwer e referable toV. fischeri. They further stated that thelea f and flower characteristics of the "twospecies " were scarcely distinguishable and that it was doubtful whether V. keniensis was distinct from V. fischeri. Verdcourt (1992), observed that V.keniensis is usually synonymised with V. fischeri but he regarded them as distinct taxa worthy of specific rank. However, the morphological characteristics such as flowers and leaves as described by Beentje (1994) and Verdcourt (1992) are in reality hardly distinguishable to rank the two species as separate entities. Appendix 2.1 compares the exact wording or lack thereof in the descriptions by the authors.
Therefore studies todetermin e the species' population size, mating systems and the genetic structure wasprecede d by investigating their taxonomy toclarif y thedistinctio n between them. It has been unclear whether the two were distinct species, ecotypes or intra-specific taxa. The geographical distribution of the species in East Africa (Figure 2.3) was studied and laboratory analysis of representative specimens from their natural populations done. Herbarium specimens and seed sampleswer ecollecte d from the four Vitex populations of Mt. Kenya, Nyambene hills, Mt. Elgonan d Lake Victoria regions for further observation and analyses. The leaf and flower specimens from the sampled
23 Figure2.3 . Natural range ofdistributio n ofV. keniensis and V.fischeri i nEas t Africa.
24 Vitex populations were kept at EA, KEFRI (Kenya) and WAG (Netherlands). Laboratory analyses were carried outt odetermin e themorphological , cytological and genetic relationships of the Vitex'spopulations . Further studies were carried out on their mating systems to generate additional information relevant to its sustainable management.
The morphological characteristics of V. keniensisan d V. fischeri (Appendix 2.1) used for keeping them apart are apparently not clearly distinct. Most of the traits used in separating them overlapped and did not vary between the two. This observation is in line with the scepticism expressed by Dale & Greenway (1961) although Beentje (1994) continued to consider the two as different taxa. It is therefore most likely that the two are not distinct as will be explained in the following chapters.
Most of the authors handling the two taxa as separate species have relied mainly on their geographical distribution. The dependence on geographical distribution and habitats as the principal taxonomic tool could be unreliable because trends and rates of evolution and speciation like most natural phenomena are unpredictable and are under the influence of a number of different factors. Despite the variation in their natural range of distribution, the close similarity between V.keniensis an d V. fischeri casted doubts on their being separated taxonomically. Trees like Vitexhav e long life cycles and this can slow down the rate of genetic drift thus delaying speciation in isolated populations. Despite the geographical and ecological separation between the different Vitexpopulations , the varying degrees of selection pressure and the limited geneflo w betweenthem , theyretaine d similar morphological features. Thisi spossibl y implying that there are other factors maintaining the similarity between the disjunct Vitexpopulations .
Some authors further relied on traits such as height, crown size and shape. This criterion has ahig h chance of leading to even adeepe r confusion. That isbecaus e the abovetrait sar e toa larg eexten t influenced byth eprevailin genvironmenta l conditions, ageo f theplan t and their assessment isnormall y subjective. Most of adaptivechanges , involvealteratio n ofgenotype s (Stebbins, 1974),an dvariatio n inheigh tan dcrow nsiz e may not necessarily imply complete genetic alteration or separation of the populations into distinct taxa.
Geographically, the four populations of Vitexar e isolated, limiting the possibility of geneflo w between themwhic hmostl y occursthroug hpollinatio n andsee ddispersal . Therefore, the morphological similarity between the Vitex population cannot be associated with inter-population gene exchange. The morphological similarity despite the geographical isolation and variable ecological conditions between the populations could be attributed to common ancestry and biological traits enabling the species' populations to withstand selection pressure in the different ecological zones.
The subsequent chapters investigate more biological characteristics that could cast more light on the taxonomy of the Vitexspecies . The study is based on additional
25 taxonomical traits: seed morphology, seedling morphology, wood properties and cytological characteristics, mating systems and genetic structure of the different Vitex populations in East Africa.
Specimens examined
KENYA: Ahenda JA 1 (WAG): Muguga-Nairobi; Ahenda JA 2 (WAG): Chuka forest; Ahenda JA 3 (WAG): Chogoria Forest; AhendaJ A 4 (KEFRI): Irangi Forest; Ahenda JA 5 (KEFRI): Nyambene hills. Mikinduri; AhendaJ A 6 (KEFRI): Kimilili; Ahenda JA 7 (KEFRI): Kimilili division; Ahenda JA 8 (WAG): Malaba; Balbo & Balbo 78 & 846 (EA); Battiscombe960 : Nandi; BattiscombeK48 4 (EA): Nyanza basin; Battiscombe K563 (EA): Southern Mt. Kenya; W.R. Buch 60/455 (EA): Isebania; W.R. Buch 61/190 (EA): Bungoma; I.R. Dale 4752 (EA): Kakamega/Kai- mosi; I.R. Dale K3125 (EA): Kakamega; R.B. Faden & H. Beentje 466 (EA): Ngangao forest (Taita hills); Fischer 476 (EA); L.P. Glasgow 46/46 (EA): Port Victoria; D.K.S. Grant 846 (EA); E.J. Humore 15669 (EA): Mt. Kenya; C.H.S. Kabuye15 2(EA) :Pangan i(Nairobi) ;/ . Karuhanga 877(EA) :Low yChurc h (Nairibi); Kenya Horticultural Soc. 1 54/263 (EA): Chogoria forest; P. Kuchar 9496 (EA): Masai Mara; P. Kuchar 11654 (EA): Masai Mara; vander Maesen& Ahenda 6196 (WAG): Meru; vander Maesen& Ahenda 620 2 (WAG); Nyambene Tea Estate; van derMaesen & Ahenda 620 3 (WAG): Lake Victoria Basin. Suba-Kuria (near Migori); vander Maesen& Ahenda 6204 (WAG): Suba-Kuria (near Migori); vander Maesen &Ahenda 6205 (WAG): Suba-Kuria (near Migori); vander Maesen & Ahenda 620 6 (WAG): Beroya-Taranganya (neaTanzania/Keny a border); van derMaesen &Ahenda 6207 (WAG): Kadongo-Nyaduong (God Jope); van der Maesen & Ahenda 6209 (WAG): Ogwedhi- Moasai; van der Maesen& Ahenda 6212 (WAG): Kamasela; J. McDonald s.n.: Songhor; McDonald 2728 (EA); J.L. Moon 15078 (EA): Nyanza basin; NRB3 9 (EA): Kitale; W.B.N. Pamba 74 (EA); Polhill& Verdcourt 275 (EA): Nyambene hills; S.A. Robertson & Q. Luke 6322 (EA): Gogoni forest (Kwale); Stuhlman357 6 (EA); Trapnell 59/62 (EA): Donyo Sabuk; Tweedie 2599 (EA); G.R. Williams 276 (EA): Kakamega; S.H. Wimbush 1936 (EA): Meru.
UGANDA:Ahenda J A 17(KEFRI) :Entebbe ;Ahenda J A 18(KEFRI) : Iganga-Jinja; AhendaJ A 19(KEFRI) : Busia; AhendaJ A 21 (KEFRI): Serere-Lake Kyoga;Ahenda JA 22 (KEFRI): Gombe-Kampala;Ahenda JA2 0 (KEFRI): Pallisa-Mbale;Ahenda J A 23 (KEFRI): Bosolwe-Tororo; C.V. Brasnett s.n.(EA): Gombolola (Lake Kyoga); P. Chandler 832 (EA): Serere (Teso);D. Dawkins 415 (EA): Mengo; W.J. Eggelings.n . (EA): Bugala-SeseIslan d (LakeVictoria) ;Morris Godall s.n . (EA): LoluiIslan d (Lake Victoria); CM. Harris219 a (EA): Jinja; A.B. KatendeK26 6 (EA): Teso-Mt. Abela; A.B. Katende 2193 (EA): Acholi-Ilamwo county; A.B. Katende 2346 (EA): East Mengo; K.A. Lye 4372 (EA): East Mengo; Milchemore 1278 (EA): Busoga; A.S. Thomas407 2 (EA): West Nile; A.S. Thomas 1222 (EA): Bugala-Sese Island (Lake Victoria); — 1580 (EA): Moroto Mts.
26 TANZANIA: Ahenda JA 9 (KEFRI): Usagara-Mwanza; Ahenda JA 10 (KEFRI): Ibondo-Mwnaza;Ahenda J A 11(KEFRI) : Musoma;Ahenda J A 12(KEFRI) : Tarime; Ahenda JA 13 (KEFRI): Shirati; Ahenda JA 14 (KEFRI): Utegi; Ahenda JA 15 (KEFRI): Buhemba;Ahenda JA 16(KEFRI) : Shinyanga; S.Azuma 47 0(EA) :Kabog o Mts (Kigoma); S. Azuma 476 (EA): Kabogo Mts (Kigoma); B.D. Burth 3294 (EA): Shinyanga; Carmichael 835 (EA): Rubya forest reserve-Ukerewe Island (Lake Victoria); T.H. Clutton-Brock s.n. (EA): Kigoma; T.H. Clutton-Brock 433 (EA): Gombe (Kigoma); E.A.H. 13, 116 (EA): Musoma; P.J. Greenway 799 (EA): Muritikera; P.J. Greenway &R.M. Polhill 11431 (EA): Manyani (Dodoma); J. Itani s.n. (EA): Kigoma; K. Kano25 3 (EA): Firabanga river (Kigoma); Z.N. Kasika805 6 (EA): Zanaki-Sukuma; Kew 336 (EA): Musoma; J. Newbould s.n. (EA): Geita- Maisamu Island (Lake Victoria); S. Paulo 53 (EA): Uluguru Mts (Morogoro); J.R.A. Procter s.n. (EA): Capri point (Mwanza); Procter98 3 (EA): Bukoba; Procter347 8 (EA): Ngurdoto crater (Arusha); V.V.Rauma 255 (EA): Capri point (Mwanza); W.A. Rogers 1516 (EA): Biharamulo; F.G. Smiths.n . (EA): Butimba (Mwanza);A. Suzuki 105 (EA): Kasakati (Kigoma);A. Suzuki12 1(EA) :Kasakat i (Kigoma);R.E.S. Tanner (EA): Musoma; TCB 348 (EA): Kigoma; Schleiben 3148 (EA): Uluguru Mts (Morogoro); S.R. Semsei360 3 (EA): Lushoto Arboretum; S.R. Semsei399 4 (EA): Magamba (Lushoto); Von Braun 1053 (EA): Dodwe Derema road.
Other related specimens examined: Mildred E. Mathias & Taylor Al 51 (EA); Peiper105 3(EA) ;Reekmans 102 5(EA) : Burundi; V.C. Gilbert 5240 (EA): Tanzania; Welwitsch 5696 (EA): Angola.
2.6 Morphological and cytological characteristics of V. keniensis and V. fischeri
Besides examining the herbarium specimens, further studies were carried out to compare and contrast the four different populations of V. keniensisan d V. fischeri in East Africa todetermin e whether thetw o species belong todifferen t or the same taxon (species). The study involved the analysis of the morphological and cytological traits, mating systems and the genetic structure of their populations.
2.6.1 Morphological variation in V. keniensis andV. fischeri seed characteristics
In many genera fruits and seeds produce taxonomic criteria at different levels of hierarchy (Stace, 1980). The seed dodiffe r inter-specifically, incertai n cases it varies even at infraspecific level (e.g. Spergula arvensis) or even within an individual (e.g. Spergularia media)(Stace , 1980). Such quantitative characters tend to be more useful at lower levels of taxonomic hierarchy (Davis & Heywood , 1963). The morphology of Vitexseed s from the different populations was evaluated and compared.
Materials and methods 1. 100tree s were marked from eacho f the V.keniensis and V.fischeri populations ;
27 Mt. Kenya, Nyambene hills, Mt. Elgon and Lake Victoria, totalling 400 trees from the four populations of the species. 2. At maturity, 10 fruits were collected from each of the sampled trees, processed (extracted and cleaned) and kept separately per mother tree. 3. The seeds from the trees of each V. keniensis and V. fischeri population were thenbulke d and 200seed s randomly drawn from the seed-lot of each population. 4. Measurements on the length and width of the randomly drawn seeds were taken using vernier callipers andth eweigh t of eachwa s seedmeasure d ona nelectroni c weighing balance.
Results and discussions The variability on seed characteristics of seeds from the different Vitex populations were evaluated based on their differences in seed length, width, shape (derived from length: width) and weight. Analyses of variance of the seed length, width, shape and weight were carried out using the SPSS computer program (Nouris, 1994) to investigate the differences among the V. keniensis and V. fischeri populations of Nyambene hills, Mt. Kenya, Mt. Elgon and Lake Victoria. Tukey-hsd tests at significance level of 0.05 in SPSS was used to separate the means.
The results of analyses of variance on the Vitexsee d morphological traits showed significant differences atp < 0.05 level among thefou r Vitex populations (Table 2.1). Thehighes t variation among the Vitex populations wasrecorde d insee d shape (derived from therati o of thesee d length towidth) . Meanwhile theleas t variation was observed in seed length. The level of variations on seed weight and seed width between the studied Vitex populations were moderate.
Overall, the Mt. Kenya Vitexpopulatio n had the longest mean seed length while Nyambene hills had the shortest (Table 2.2). There was no significant (p < 0.05) difference between Nyambene hills, Mt. Elgon and Lake Victoria populations in the means of the lengths of their seeds. The Lake Victoria population of the species produced thewides t seed and Mt. Elgon, the narrowest. Themea n seed width of Lake Victoria population did not differ (p < 0.05) significantly from those of Nyambene hills and Mt. Kenya. Lake Victoria Vitex produced lighter and more spherical seeds while Mt. Kenya Vitexseed s were the heaviest and most elongated in shape.
Conclusion Most of the variation observed in the V.keniensis and V.fischeri see d morphologi cal characteristics did not seem to follow any geographical or ecological pattern. No clear pattern of the distribution of the seed morphology was observed among the populations. Assuming theobserve d seed morphological variation isa reflection of the species' genetic make-up, then the random distribution of most seed characters could be a result of lack of difference between the Vitex populations; possibly a result of the common genetic background of the Vitex populations or because of their capacity to survive the different ecological conditions without conspicuous morphological change in their seeds.
28 Table 2.1. Analysis of variance of seed length (cm), seed width (cm), Seed shape (length : breadth) and seed weight (g) in the four V. keniensis and V. fischeri populations; Mt. Kenya, Nyambene hills, Mt. Elgon and Lake Victoria. (* denotes significance at the 5% level, ns denotes non-significance at 5% level).
Source of DF Length Width Shape Weight variation MS F MS F MS F MS F
Population 3 0.49 8.62* 0.36 17.66 1.37 31.30* 0.59 14.35* Replication 3 0.40 7.06* 0.17 8.51* 0.02 0.49ns 0.66 16.07* Residual 793 0.06 0.02 0.04 0.04
Table 2.2. Mean seed length, width, seed shape (length : width ratio) and weight of the Vitex seeds from the four different Vitex populations; Mt. Kenya, Nyambene hills, Mt. Elgon and Lake Victoria (Means denoted by the same letter are not significantly different at the 5% level of significance according to Tukey test).
Vitex Populations Length (cm) Width (cm) Shape Weight (g)
Mt. Kenya 1.3475 b 0.8570 b 1.5905 b 0.5535 c Nyambene hills 1.2445 a 0.8655 b 1.4502 a 0.5299 be Mt. Elgon 1.2460 a 0.7890 a 1.5751 b 0.4888 b Lake Victoria 1.2575 a 0.8875 b 1.4305 a 0.4292 a
However, it is also possible that most of the seed traits are selectively neutral and therefore do not respond to the forces of natural selection. That may make them neither adaptive nor sensitive to changes in the environmental conditions thus leading to the observed random distribution of seed morphological traits among the different populations. Despite the significant variation (p < 0.05) between Vitex populations on seedmorphology , littlelin kwa sobserve d inrelatio nt oth eobviou sdifferen t ecological conditions of the different habitats of the species. Therefore, basing on the seed morphology no consistent distinction can be made between the populations to support their separation intoV. keniensis and V.fischeri. However , afurthe r studywa s carried out on other morphological characteristics of the different populations of the species and are presented in the next sections.
2.6.2 Seedling leaf morphology of V. keniensis and V. fischeri
Materials and methods 1. In each of the four V. keniensis and V. fischeri populations 50 distantly distributed trees were randomly sampled. 2. One seedling was raised from each of the sampled trees making a total of 50 seedlings per Vitex population. 3. The 50seedling s per Vitexpopulatio n were raised inlabelle d potsunde r thesam e conditions in a glasshouse in 10 replicates of 5 seedlings.
29 4. At the age of three months when the seedlings started producing compound (palmate; 5-foliolate) leaves, the leaf length, width, and petiole length were measured usingvernie r callipers andth enumbe r of simpleleaves , veins andteet h (serration) counted.
Results and discussions Analyses of variance were carried out on the seedling leaf morphology using the SPSS computer program (Nouris, 1994) to investigate the differences among the four Vitex populations. Tukey's-hsd tests at significance level of 0.05 in SPSS was used to separate the means of the seedling leaf morphological traits.
The analyses of variance on leaf morphology of seedlings from the different populations of Vitexshowe d significant (p < 0.05) differences between them (Table 2.3). The seedlings were raised under similar environmental conditions in a glasshouse, therefore any differences among them should be a reflection of their taxonomic andgeneti cvariation . Thehighes t significant variationamon gth efou rVitex populations wasobserve d inseedlin g leaf length, width andnumbe r of veins. Theleas t significant variation was observed in the number of teeth (serration). The within population variation in the seedling leaf morphology at p < 0.05 was insignificant except for the number of teeth (serration). That could be a result of similar genetic background and slow pace of population divergence.
Table2.3 . Analysis of Variance onLea f length (cm), width (cm), petiole length(cm) , number of simple leaves, veins and number of teeth of seedlings of V. keniensisan d V.fischeri fro m Mt. Kenya, Nyambene hills, Mt. Elgon and Lake Victoria (*denote s significance at 5% level and ns non-significance at 5% level).
Source of DF Leaf length Leaf width Petiole le ngth variation MS F MS F MS F
Population 3 21.80 12.38* 5.44 11.39* 0.73 4.12* Pop.*Tree 12 1.22 0.70ns 0.48 1.01ns 0.24 1.34ns Replication 9 2.38 1.37ns 0.57 1.20ns 0.89 4.98* Residual 175 1.74 0.48 0.18
Source of DF No. of te 9th No. of sir nple No. of ve ins variation leaves
MS F MS F MS F
Population 3 8.47 3.25* 5.77 5.79* 396.82 14.25* Pop.*Tree 12 1.56 0.60* 0.85 0.86ns 37.04 1.33ns Replication 9 5.58 2.14ns 3.14 3.16* 43.26 1.55ns Residual 175 2.60 1.00 27.84
30 Table 2.4. Means of length, width, petiole length, number of teeth, simple leaves and veins of Vitexseedling s from the four different V. keniensis and V. fischeri popula tions; Mt. Kenya, Nyambene hills, Mt. Elgon and Lake Victoria, (means denoted by the same letter are not significantly different at 5% significance level according to Tukey test). VitexPopulatio n Leaf length, cm Leaf width, cm Petiole length, cm Mt. Kenya 5.517b 3.140b 1.154b Nyambene hills 4.491a 2.534a 0.970ab Mt. Elgon 4.057a 2.375a 0.888a L. Victoria 4.219a 2.711a 0.908a Vitexpopulatio n No. of teeth No. of simple No. of veins leaves Mt. Kenya 9.480b 4.100ab 26.940c Nyambene hills 9.000ab 4.300b 33.820a Mt. Elgon 9.460ab 3.820a 30.840b L. Victoria 8.620a 3.520a 30.500b
On multiple range tests (Tukey-HSD test with significance levelp < 0.05), the four Vitex populations exhibited no clear pattern distinction (Table 2.4). Seedlings of Mt. Kenya and Nyambene hills Vitex differed significantly (p < 0.05) on most of the leaf morphological traits despite their geographical proximity and ecological similarity of their habitats. The same observation was made on the seedlings of Lake Victoria and Mt. Elgon. The pattern of distribution of most of the variation in the seedling leaf morphological traits were also inconsistent with the expected ecological gradient and geographical distances. The seedlings raised under the similar growthcondition s were expected to exhibit their heritable (adaptive) traits. Therefore, the lack of distinct pattern of variation inseedlin g leaf morphology of thedifferen t Vitexpopulation s seem to imply similarity between them.
2.6.3 Wood characteristics of V. keniensis and V. fischeri
Wood anatomy is of considerable taxonomic and phylogenetic significance as cited by Carlquist (1961), besides timber trade. There area numbe r of features of thexyle m which show major trends of evolution in angiosperms. Distribution of vessels in transection is ataxonomicall y significant feature (Singh et al., 1994). The presence or absence of tylose and presence of visible pits in tylose also afford taxonomic criteria. Other features include vascular rays and storied wood (Stace, 1980). The study on the properties of wood of Vitexfro m the different populations was carried out to make comparisons between them. The results can also be used in the subsequent selection and genetic improvement of the species for commercial timber production.
31 Materials and methods The characteristics of V. keniensis and V. fischeri wood from the different populations have been evaluated by analyzing samples from the four different populations of the species. That was an additional basis for assessing the distinction and similarity between the two "species".
1. 10piece s of wood were collected from each of the Vitex populations. The wood samples were collected from mature trees of approximately the same age. 2. The specific gravity of each sample was measured as a ratio of the weight of a sample of wood to its volume. 3. Threemajo r planeswer euse d for describing themicro -an dmacro-feature s ofth e Vitexwood . The transverse, radial and tangential sections of the wood samples were studied. In the transverse section, planes were made perpendicular to the logs; radial sections, vertical planes were cut through the central axis of the logs parallel to the rays, tangential sections, parallel vertical planes but not through the longitudinal axis (Figure 2.4). 4. Woodhardines s inkilonewto n andfibr e lengthi nmillimeter s (mm) ofwoo d from the different Vitex populations were analyzed.
Results and discussions The average wood density from thedifferen t Vitex populations varied between 0.42 and 0.51 g/cm3. The mean values for Mt. Kenya, Nyambene hills, Mt. Elgon and Lake Victoria were 0.42, 0.45, 0.48 and 0.51 g/cm3 respectively. The density of the sampled Vitex wood wasmoderate , falling betweenthos e of hard wood and soft wood. Soft woods growing in East Africa are Pinus radiata (0.33 g/cm3), Pinusoocarpa (0.441 g/m3) and P.patula (0.33t o0.4 6 cm3). Hardwood includesAcacia Senegal (L.) Wild. (0.54 cm3) and Acacia nilotica(L. ) Del. (0.94 cm3) (Giertz, 1995). The mean density of about 0.45g/cm3 in Vitex would therefore be treated as medium in the classification by Wheeler et al. (1989). The difference between the different populations of Vitexi n wood density ranged from 7 to 21%. The largest difference (21%) was observed between Mt. Kenya and Lake Victoria. The Vitexwoo d density seems to follow some consistent pattern in relation to the ecological and geographical ranges. However, at this stage, it is difficult to conclude whether the traits are heritable or not.
Transverse section (xlOO): Thepore s are solitary and theparenchym a vasi-centric. Some parenchyma were paratracheal or absent. The rays are predominantly multiserrate (2 to 4 cells wide) and the fibres septate. The fibres are thin to thick walled, growth rings indistinct and tylose present. Radial section (xlOO): Ray tissues are homogeneous composed wholly of procumbent cells. Tangential section (xlOO): The rays are 1 to 2cell s wide but predominantly uniseriate, fibres septate, one septum per fibre (Figure 2.4).
The mean radial hardiness of Vitex wood was between 1.87 and 2.62 kilonewton, while the tangential hardiness ranged from 1.94 to 3.20 kilonewton. The average
32 mmw- russetST" '
i!!* • "Er 1mm
Figure 2.4. Morphological properties of wood of V. keniensis. a)Transvers e section,b )Tangentia l section, c) Radial section (xlOO).
33 E E B Mt. Kenya O) c B Nyambene § Mt. Elgon HL .Victori a c to CD
Outer Inner Outer Inner sapwood sapwood heartwood heartwood
Figure2.5 . Mean fibre length of V. keniensisan d V.fischeri wood . hardiness of Vitex wood varied among the populations. The wood from Lake Victoria and Mt. Elgon had higher values of 2.62 and 2.5 kilonewton (radial hardiness) respectively. Lake Victoria had tangential hardiness of 2.54 and Mt. Elgon, 3.20 kilonewton. The mean hardiness of Vitex from Mt. Kenya was 1.87 (radial hardiness) and 1.97 kilonewton (tangential hardiness). Meanwhile, Nyambene hills had radial hardiness of 1.99 and tangential hardiness of 1.94 kilonewton. Thepropertie s ofwoo d from Lake Victoria and Mt. Elgon seem to exhibit stronger physical properties than the timber of Mt. Kenya and Nyambene hills. The strong physical properties in wood are preferred in construction, therefore it could be one of the desirable traits for improvement breeding in forestry.
The heartwood of Vitex is dark brown in colour and the sapwood is light brown to yellow. The lighter colour of sap wood is a result of it being young, living and physiologically active. The heart wood is normally darker because of deposits of resins, tannin and other organic substances. They make the timber resistant to fungi and insects. The grains are straight especially inthos e from Nyambene and Mt. Kenya but slightly wavy and interlocked inthos eo fLak eVictori a andMt . Elgon. Thetextur e is fine but relatively coarse among those from Lake Victoria and Mt. Elgon. Mt. Kenya Vitex had the longest fibres and Lake Victoria the shortest. The Vitex populations of Mt. Kenya and Nyambene hills had the longest mean fibre length (Figure 2.5). The wood from the different Vitexpopulation s varied in their physical properties (density, hardiness and fibre length) but not in their micro- and macro- morphological characteristics.
There is a strong correlation between wood structure and macro-ecology of tree species (Soerianegara & Lemmens, 1993). Therefore, it is not expedient to separate
34 the different populations into taxonomic entities based on the wood density, hardiness and fibre length while the other morphological traits and genetic characteristics are similar. The variation in the physical properties of wood can also arise from the influence of the environment through phenotypic plasticity without necessarily involving of genetic change (Stuessy, 1990).
2.6.4 Cytological characteristics of V. keniensis and V. fischeri
Cytology in a broad sense deals with all aspects of cells, but in practice in taxonomic work the focus has been on chromosome number and their attributes (Lewis, 1969). Chromosomes play a special role as a source of comparative data because they contain genetic material responsible for maintaining biological integrity of species. Chromosome number as ataxonomi c character isprobabl y oneo f the most constant single features employed (Singh et al., 1994). Generally, with a few exceptions, thenumbe r of chromosomes ineac hcel l of individuals of thesam e species is normally constant (Stace, 1980; Singh et al., 1994). The relative conservativeness renders the chromosome number an important and much used taxonomic character (Stace, 1980). Since chromosome numbers change in evolution through many diverse avenues, comparisons arenormall y validonl ywithi ncertai nnarro wdefine d limitssuc h as species within a genus (Stace, 1980).
The ploidy level effects a number of characteristics in plant life for instance, the morphology, physiology and even the mating systems (Stace, 1980). The distribution of cytotypes in species populations cantherefor e beuse d todistinguis h them andmak e some conclusions on their taxonomic relationships. Studies on the cytological characteristics of V. keniensis and V. fischeri were carried out on the different populations of the species. The information on the distribution of the cytotypes was also essential for interpreting complex electrophoretic data generated from genetic (isozyme) analysis onth especies 'population s (Barrington, 1990;Haufle r etal. , 1990), in chapter 4.
Materials and methods 1. 10matur e trees were sampled from each of the four Vitex populations in shown in Figure 2.3. 2. Mature fruits were collected from the sampled trees and the seeds extracted, cleaned and germinated in vermiculite in labelled petri-dishes placed in an incubator set at 25°C . 3. Once the seeds germinated and produced radicles, 2 to 3cm of the meristematic root tips were cut off and immersed in 2 mM 8-hydroxyquinoline at room temperature for 4 to 7 hours. 4. Thepre-treate d root tipswer e fixed in freshly prepared aceticaci d toalcoho l (1:3 parts v/v glacial acetic acid to absolute alcohol), one to two hours. 5. The root tips were hydrolysed in a normal solution of hydrochloric acid in a water bath at 60°C for 8-12 minutes, then stained with Feulgen reagent in the
35 dark at room temperature for about one hour. 6. The stained root tips were then squashed in 2% acetocarmine solution and observed under a microscope. 7. 10 cells at metaphase stage with clearly visible chromosomes were counted in each slide and the number of chromosomes in each of them determined.
Results and discussions The best samples of root-tips i.e. those with actively dividing cells were obtained from the fast growing seedlings.Therefore, the growth rates of the plants had direct influence on the possibility of getting cells with visible chromosomes at metaphase stage. The time of sample collection also had an influenced on the chances of getting good root-tips. The best root samples were harvested at noon when it was bright and warm, that isprobabl y whenth e meristematic cells in the roots wereundergoin g rapid division. The roots collected in the morning or late in the evening had very low proportion of actively dividing cells. Root pruning and withholding watering of the seedlings for 24 hours before collecting root-tips also enhanced root growth.
However, the resultant chromosomes of Vitex were too tiny for their morphology to be distinguishable for detailed studies on the nature of their karyotypes. The chromosome counts from the randomly selected 10 trees of each of the four tree populations showed that the species had 96 somatic chromosomes (Figure 2.6). This figure was constant and applied to all the samples from the four different Vitextre e populations. Somatic chromosome numbers reported in the genus Vitex sofa r vary for 24 to 34. Some of the species in the genus have been reported to have more than one cytotypes; V. agnus-castus L. 2n=24 (Patermann, 1935) and 2n=32 (Sharma & Mukhopadhyay, 1963); V.trifolia L. 2n=26 (Sobti &Singh , 1961), 2n=32 (Suguira, 1936) and 2n=34 (Sharma & Mukhopadhyay, 1963); V. negundoL . 2n=24 (Malik & Ahmad, 1963), 2n=26 (Sobti & Singh, 1961) and 2n=34 (Sharma & Mukho- pandyay, 1963). But only one cytotype has been reported on the following Vitex species; V. cienkowskii Kotschy et Peyr. 2n=32 (Mangenot & Mangenot, 1962), V. fosteri Wright 2n=32 (Mangenot & Mangenot, 1962), V. grandifoliaGiirk e 2n=32 (Mangenot &Mangenot , 1957), V.micrantha 2n=32 (Mangenot &Mangenot , 1957) and V. rotundifolia 2n=32 (Chuang et al., 1963). Inth e past, two basic chromosome numbers of x=6 and 8hav e been reported for the genus Vitex(Darlingto n & Wylie, 1956). The chromosomes in the meiotic cells of V. keniensis and V.fischeri wer e too small to beobserve d son oconclusio n could made onthei r basic chromosome number. Recently, isozyme andothe r molecular techniques havebee nuse d todetermin e ploidy level in plants (Crawford & Smith, 1984; Haufler et al., 1990; Harris & Ingram, 1992). Inferences on the nature and level of polyploidy in the species were therefore made from their isozyme phenotypes (chapter 4).
The Vitexspecie s is therefore highly polyploid. The somatic chromosome number was the same in all the populations studied i.e. 2n=96. Polyploidy has long been recognised as a significant feature in many plant taxa and estimates vary, but 47 to 70% of all angiosperm species have polyploid origins (Masterson, 1994). Polyploids
36 •*xr2 v* • v
A#/vV* -> Figure 2.6. Somatic chromosomes of V. keniensisan d V. fischeri. a) Mt. Kenya, b) Nyambene hills, c) Mt. Elgon, d) Lake Victoria (x 1000).
frequently have long ecological ranges different from their diploid progenitors (Ehrendorfer et al., 1996; Lewis, 1980). This attribute could be linked to the wide range of chromosomal combination possibilities in a polyploid. A number of studies have attempted to show the relationship of polyploidy to the environment. Past research has shown higher ecological tolerance in polyploid than the closely related diploids (Stebbins, 1957). Polyploidy therefore seem to confer additional ecological adaptability toman ytax amakin gthe mcapabl eo fcolonisin g awid egeographica l range (Stuessy, 1990). Therefore, the observed high polyploidy in Vitex could partly explain theabilit y of the species to occur inth econtrastin g ecological zones without changing its morphological and genetic characteristics.
According to Stuessy (1990), variation in the number of chromosomes is more common in herbaceous than in woody dicots. Therefore many of the woody species
37 tend to exhibit a uniform chromosome number despite ecological diversity as in Danthania sericea (Fairbrothers & Quinn, 1970). The constant number of somatic chromosomes throughout the Vitex populations indicates that despite the variable ecological conditions, the species has retained one cytotype. The observation further underlines the similarity between V. keniensisan d V. fischeri, casting doubt on their distinction. The close connection between chromosomes and reproductive isolation makes it logicalt oconside r thedifferenc e inchromosom enumbe r betweenpopulation s to be recognised as taxonomic at specific level. The chromosomal traits do reflect common ancestry rather than convergence (Stuessy, 1990), implying that theobserve d similarity in the cytotype of the Vitexpopulation s might arose from their common origin especially considering theclos e similarity inthei rphenotypi c traits as illustrated in the previous sections in this chapter.
2.7 Conclusion
The studies of the herbarium specimens, seed and seedling morphology, somatic chromosome number and wood properties of Vitex revealed more similarities than distinction between the populations from the different ecological areas. The patterns of variation in most of their morphological features were random, implying that V. keniensisan d V. fischeri are not taxonomically different from each other.
In nature there are three possible ways through which such disjunct populations of species are formed (Lind &Morrison , 1974);the yma yoriginat e separately in different areas simultaneously (multiple origin), they may be formed by long distance dispersal from one to another climatically favourable area, or they may occur through contraction or separation of a once continuous range following climatic change or geological events or human activities. The first postulation isunlikel y especially under natural conditions. The second postulation may be ruled out because of the vast distances between the Vitex populations thus limiting the inter-population geneflow.
It is most probable that the Vitex populations were formed through the third postulation as a result of various geological and tectonic activities in East African. Biogeographical history study ofEas tAfrica n vegetation indicated thatdurin g theearl y part of the Kainozoic Era East Africa had no high mountains and is believed to have consisted of a vast gently undulating plain (Lind & Morrison, 1974) probably consisting of similar vegetation cover. The present highlands are therefore a result of tectonic activities in the Miocene and Pleistocene which led to the formation of Rift Valley and volcanic mountains (Haughton, 1963; Lind & Morrison, 1974). It is therefore possible that most of the plant species were separated into different populations by the geological events and human activities that followed.
The biogeographical history could also explain the relict of highland species such as Podocarpus latifolia(Thunb. ) Mirb., Prunus africana Mill., Oleaeuropea L. ssp. africana (Mill.) P. Green and Warburgia ugandensis Sprague found insom e low-lying
38 areas in the wet savanna around Lake Victoria at about 1000 to 1400 m above sea level. These species have been considered to be exclusively sub-montane or montane forest plants. The western shore of Lake Victoria in Maramagambo forest (Uganda) and Minziro forest (Tanzania) at about 1000 to 1200 m above sea level is dominated by Podocarpus species (Lind & Morrison, 1974). The natural occurrence of these species in this region is considered remarkable because this is far below their known natural altitudinal range. Meanwhile, in somepart s of thewe t savanna of Masai Mara, Migori and Kuria districts of Kenya Vitex do occur naturally inth e same zone with P. africana,O. europea ssp.africana andW. ugandensisespeciall y insom eriverin e areas where human activities are minimal. InMorot o mountains inUgand a also, the species grows in association with O. europea and Tecleanobilis Del . The distribution of V. keniensis and V. fischeri in the different geographical and ecological zones in East Africa can be explained by the biogeographical historical events which led to the formation of the Rift Valley and volcanic mountains separating the once continuous lowland vegetation stretching from West to East Africa.
The descent from common ancestry could therefore explain the morphological similarity observed in the populations of V. keniensisan d V. fischeri occurring in the different geographical and even ecological regions of East Africa. In addition to common ancestry, the morphological similarity could have maintained by slow pace divergence between the isolated populations caused by the species' biological characteristics such as polyploidy, long life cycle (over 100 years) and phenotypic plasticity. These factors can slow down the rate of adaptive changes in geographically and ecologically isolated populations.
Alternatively, the difference in selection pressure between the regions may not be as much as it appears. Therefore the main morphological and physiological features of the species may be adequate for its populations to colonise the distinct ecological zones without the need for adaptive changes. The results of this study therefore indicates that the role of geographical and ecological separation in speciation was overestimated for the Vitex populations. The study has further indicated that geographical and ecological separation of populations of a species alone is inadequate for taxonomic separation unless supported by other factors, otherwise the ecophenes would end up in an endless species list.
The stability of Vitexove r a wide ecological range could be an indication that so long as the morphological, physiological and genetic make-up of a species does not impose limitations on its survival, there would be less need for adaptive change thus divergence would be unnecessary or it proceeds at a slow pace. However, over time and as the natural environment changes and pressure from human activities increase, thespecies ' populations mighteventuall yunderg o speciation events.Bu ta tth emomen t the observed morphological and cytological variations are not adequate for them tob e placed in separate taxa. V.keniensis andV. fischeri should therefore be considered as a single species. This isals osupporte d by the observations madeb y Dale & Greenway (1961).
39 V. fischeri Giirke in Engl. Bot. Jahrb. 18: 171 (1893). Types:Tanzania : Mwanzadistrict ,nea rLak eVictoria , Kayenzi[Kahegi] , Fischer476. (B, all syntypes destroyed). Neotype: vander Maesen& Ahenda: 6203; Suba-Kuria (Lake Victoria basin). Paratypes: van der Maesen &Ahenda; 6204 , 6205 (also Suba- Kuria).
Heterotypic synonyms: V. andongensis Bak. in Fl. Trop. Afr. 5: 329 (1900). Type: Angola, Pungo Andongo, Welwitsch 5696 (K, holo; BM, iso, not seen)
V. bequartii De Wild, in F.R. 13: 142 (1914). Type: Zaire, Lubumbashi [Elisabethville], Bequaert 319 & Homble 202 (BR, hoi.)
V. keniensis Turrill (syn. V. balbi Chiov.). in K.B. 1915: 47 (1915). Type: N.E. & E. Mt. Kenya, probably Meru, D.K.S. Grant in F.D. 846 (K, syn.!, E.A., Isosyn.!). This does not qualify as a synonymusnovus, as in the past the species have been considered conspecific on and off.
V.payos (Lour.) Merr. var. stipitataMold , in Phytologia 8: 72 (1961). Type: Uganda, Mengo district, Kampala, E.H. Wilson 194 (UC, holo.!)
The name V.fischeri ha s priority over the well-known V.keniensis (Mer u oak), so V. keniensis falls into synonymy, thus V. fischeri Giirke (syn. V. keniensis Turrill). The four populations should be considered as one species and be included in the proposed conservation or improvement breeding strategies. The subsequent chapters therefore treatV. keniensis andV. fischeri a son especies ; V.fischeri syn . V. keniensis. However, both names continues tob euse d inth etex t because further comparisons are being made on their mating systems and genetic structure, and for ease of understand ing the subsequent chapters by those used to V. keniensisfo r so long a time.
40 3 Mating systems of V. keniensis and V. fischeri
3.1 Introduction
Mating systems inbroa d sense include all aspects of sex expression inplant s which affect the relative genetic contribution to the next generation of individual within a species (Wyatt, 1983). The mating system of plants is one of the major aspects that influences their population structures, and hence their genetic diversity (Loveless & Hamrick, 1984; Hamrick, 1992). The ability of any organism to produce genetic variation for any trait including resistance to pathogens, is partially determined by its mating system; self-fertilisation yields little variation in progeny and out-crossing among divergent genomes creates greater genetic variability (Bell, 1983). Generally, high genetic variability have been observed within populations of many tree species indicating that the out-crossing is common in them (Hamrick & Loveless, 1988; Loveless, 1992; Bawa, 1992). Some species are intolerant to self-fertilisation and do counter it by developing either pre- or post-zygotic rejection of selfing (Lande & Shemske, 1985; Charlesworth & Charlesworth, 1987).
The mating system of a species (such as selfing rates) may vary along geographical or ecological gradients especially under stress (Dafni, 1992). Hence, the result from onepopulatio n cannot always be applied toth e whole species. Therefore inthi s study, the four Vitex populations were treated separately. Generally, plants exhibit five main classes of mating patterns, namely: predominantly self-fertilising, predominantly outcrossing, mixed selfing and outcrossing, apomixis (Richards, 1986; Brown, 1989; Brown et al., 1989).Withi npopulation s of selfing plants, variability isusuall y lowbu t not depauperate, owing to limited outcrossing and segregation in the progeny of the few heterozygous maternal plants available (Allard, 1989; Shore, 1991).
Mating systems comprises of the mode of pollination, fertilisation, the method of reproduction and seed dispersal mechanism (Burley et al., 1986; Richards, 1986). It is widely used in agriculture and forestry to regulate and canalise the components of fecundity for selection purposes in cultivated plants. The knowledge of the sexual systems isa nessentia l background for evaluation of thedependenc e of seed production on pollination rate and type, towards the understanding of the mechanisms of gene flow within and between populations. The information is crucial in breeding programmes where effective isolation of seed orchards and in developing seed collection strategies. Mating systems has a direct influence on genetic structure of a population and therefore is essential in sampling parental trees for breeding and gene conservation, designingmultiplicatio npopulation s sucha ssee dorchard san dt oachiev e the intended packaging of required genetic traits in progeny (Adams & Jolly, 1980; Ritland & El-Kassaby, 1985; Adams, 1992).
41 Mating systems i.e. the extent to which progenies are produced from self- fertilisation or outcrossing, vary considerably among plant species and among populations (Glover & Barrett, 1986). Therefore, the understanding of a species' mating system could also be of value to unravel its taxonomic complexities and evolutionary pathways (Stace, 1980). Certain reproductive traits such as dioecy and gynodioecy, have been interpreted as a way of avoiding self-fertilisation (Darwin, 1877; Faegri & van Pijl, 1979; Lloyd, 1975; Charlesworth & Charlesworth, 1979). Despite the benefits of outbreeding, selfing has evolved repeatedly from outcrossing taxa (Stebbins, 1957;Raven , 1976)possibl y through reproductive assurance of selfing under conditions of low population density or pollinator uncertainty (Barrett, 1988).
Generally, the knowledge of the biology of tropical forests and, in particular the reproductive biology of their woody species is very limited (NRC, 1991). The reproductive syndrome ofplant s isa nimportan t facet of their adaptive responses. Like most ofth e tropical forest tree species, thematin g system of Vitexha s notbee n studied and istherefor e unknown. This is abottlenec k to their conservation and improvement breeding programmes. However, different approaches have been used to gain an understanding of mating systems in plants and estimate parameters which describe mating systems. The most conventional method used iscontrolle d pollination to assess self-compatibility and produce full-sibs (Ellis & Sedgeley, 1992; Perez-Nasser et al., 1993). I therefore carried out studies on floral morphology, phenological patterns and the mode of pollination to determine the mating systems of the different Vitex populations.
3.2 Floral morphology and phenology of V. keniensis and V. fischeri
Angiosperms haveflower s of diverse size, shapean dcolou rdependin g onth etaxon . Flowers are functional organs whose structural complexities are presumably adapted to sexual reproduction. The flower structure, phenology and evolutionary ecology of pollinationpartnership s are sointerwove n that systematists alsorel y on floral structure for identification and phylogenetic studies. The reproductive organ is considered a conservative character and iswidel y used inclassificatio n atdifferen t taxonomic levels (Stace, 1980).
The traits of flowers play an important role in controlling pollen flow within and between flowers, and withinan d betweenplants . The outcrossing and selfing levelso f a given plant species shows a tight association between the mating system, the floral size, the temporary separation of anther dehiscence and stigma receptivity, and the spatial relationship of the stigma and anther (Cruden, 1977). The level of within and between population genetic variability and hence the evolutionary potential and niche width are reliant on floral traits and pollination strategies. Every conceivable facet of the variation of inflorescence, bracts, receptacle, calyx, corolla, stamens and ovules is valuable in plant classification at different taxonomic levels (Stace, 1980).
42 The temporal and spatial separation of the male and female functions of flowers in plants has traditionally beentreate d as amean s to avoid self-pollination (Faegri &va n der Pijl, 1979). Morphological analysis of a given flower and knowledge on its temporal pattern of development are essential and can be used for determining the species' mode of pollination. The male and the female functions of the flower may mature simultaneously (homogamy), the male (pollen) may be available before the female part (stigma) is receptive (protogyny) orth estigm a mayb ereceptiv e before the pollen grains mature (protandry). These have implications on pollination and the subsequent gene flow in a given population. The development of stigma and pollen grains can be checked in vivo or in vitro. Studies were therefore carried out on the flowers to determine their mode of pollination in populations of Vitex by monitoring phenological patterns of the different populations of the species, observing floral morphology, and trends in the maturation of both stigma and pollen grains.
Materials and methods 1. Ten trees were randomly sampled in each of the four Vitex populations. 2. 25 flower buds were selected from different parts of the crown and tagged with plastic identification markers in each tree, immediately before the flowering season as flower buds were forming. 3. The number of opened inflorescence buds were counted every week on each tagged individual tree once per week from January onwards in Mt. Kenya and Nyambene hills but from December in Mt. Elgon and Lake Victoria. 4. Finally, the number of fruits formed by the tagged flowers were also counted every week as they mature. 5. The morphology of the flowers was observed using magnifying lenses and comparison were made at different stages of development.
Results and discussions The flowers are brightly coloured, scented and hermaphroditic, that isbearin g both male and female parts (Figure 2.2). The stamens are slightly longer thanth e style, i.e. it is a thrum. The floral parts of the Vitexstudie d were similar in all the populations observed. The inflorescence is composed of flowers which vary in size from 6 to 8 mm long. The four calyx lobes are green. The corolla is bilabiate with 2 small, white lobes and one large mauve, lower lobe. The bright colour of the flowers and scent are intended to increase floral display to attract more pollinators to the inflorescence (Klinkhamer &d e Jong, 1990)an d hence increasing the chances of pollination and the possibility of fruit set. The flowers are scented at maturity and it is quite certain that the species ispollinate d bybee s (Apis mellifera L.). Thebee strappe d from the flowers using insect traps and observed under a microscope were found to havepolle n grains of Vitex whenexamined . Other flower visitors included waspsan d spiders but they did not have any pollen grains. They appeared to prey on the bees and other insects visiting the flowers. The local inhabitants reported that the species was popular in honey production and that honey from its flowers was of superior quality, fetching high prices in the market.
43 160
a. o B Mt. Kenya E Nyambene S Mt. Elgon E c B L. Victoria c ra a)
December January February March
Figure 3.1. Flower phenology of populations V. keniensisan d V.fischeri.
The androecium consists of 5 stamens, the anthers are club-shaped, slightly above the level of the fork-shaped stigma. The anthers bear sticky and yellow-orange pollen atmaturity . The gynoecium consists of awhite , superior ovary witha slende r stylean d fork-shaped stigma. The ovary hasfou r loculi, bearing upt ofou r ovules. Eventhoug h the ovules are four in number, only one gets fertilised and produces only one viable seed while the rest (three) are normally aborted. The cause could be investigated further. However, this phenomenon has been attributed to zygotic competition in developing fruits (Bawa, 1990). It has also been explained by Mazer (1987) as a manifestation of sexual selection atpost-fertilisatio n stage, afeatur e common inmulti - ovuled species.
The Vitextree s aredeciduous , shedding their leavesannually . Thelea f fall coincides with fruit maturity. The shedding of leaves and mature fruits occur during the short rains between September and November. The shedding of leaves is normally followed by theformatio n of leafbuds , leavesan d later flower buds inDecembe r (inMt . Kenya and Nyambene hills) or in November (in Lake Victoria and Mt. Elgon). Flower longevity varied considerably betweenth e Vitexpopulations . Theflower s are ready for pollination between January and March in Mt. Kenya and Nyambene hills with the peak season at the end of February. But the floral longevity of each individual flower varied between 15an d 25 days, before wilting, drying and dropping of the corolla. In Lake Victoria and Mt. Elgon regions flowering occurs between December and February with the peak mid January (Figure 3.1 & 3.2). The floral longevity tended to be shorter in the Lake Victoria and Mt. Elgon Vitexpopulation s (10 to 20 days). The duration of flower persistence onth eplan t after pollination appeared todepen d on the prevailing climatic conditions (temperature and humidity). Similar observations were also made by Primack (1980). The flower longevity could contribute to the relatively prolonged duration of flower anthesis, increasing the chances of pollination.
44
June July Aug. Sept. Oct. Nov. Figure 3.2. Fruit phenology of populations of V. keniensisan d V. fischeri.
The nectar of the tree species is produced at the bottom of the flower and the production of nectar persists for about 8t o 10days . The prolonged duration of nectar production may be intended to attract multiple visits from the pollinators mainly Apis melliferaL . (bees) thus increasing the chances of pollination. The stigma becomes sticky at the receptive stage which more or less coincides with pollen dehiscence, implying homogamy, anadaptatio n considered topromot e selfing. Flowers are mostly shed and pollination complete before the onset of the long rains which start in early April. The rain dislodges the shrivelled corolla of the pollinated flowers leaving developing fruits. The late flowers are also damaged by the rains and the winds that accompany them.
In Mt. Kenya and Nyambene hills the fruits mature between September and November, while inLak e Victoria and Mt. Elgonthe y were ready earlier, that is from July to September. The ripepulp y fruits fall toth e forest floor together with dry leaves from the crowns. The fruits are mostly dispersed by hornbill birds, monkeys and even human beings who feed on the sweet pulp and discard the nuts containing seeds. The fruit and leaf fall is enhanced by the wet (rainy) and windy conditions during that period. The extracted seeds germinate readily anddevelo p intotree s especially inarea s where the forest isopene d up. The fruits that fall by gravity directly to the forest floor without having been depulped get attacked heavily by pests and fungi lowering the chances of germination. This interferes with the natural regeneration of the species in the forest. Most of thesepredator s are attracted by the sweet (nutritious) pulpcoverin g the nut. However, the predation by insects and fungi was less severe in the wet savanna populations of Lake Victoria and Mt. Elgon. That could be attributed to the sparse crown cover and thus, the exposure of the forest floor.
45 The morphology of flowers of the different Vitexpopulation s showed no marked distinction between V. fiscfieri and V. keniensis. The phenological activities of the different populations were similar. However, the timing and the duration tended to follow thepatter n of the environmental conditions. Therefore thelon g period between flowering and fruit maturity observed in Vitex of Mt. Kenya and Nyambene hills may be attributed to the cool environment caused by the high altitude and rainfall in the region. The hermaphrodity and homogamy of the flowers indicate the possibility of selfing in theplan t species. The colourful corolla, sweet scent and sticky pollen grains is an indication that the species is zoophilous and is pollinated mainly by bees (A. mellifera). That was implied from the presence of pollen grains on the bees trapped from the species' flowers. There was also a conspicuous increase in the population of the insect when the Vitex populations are on flower. The fruits of the tree species are pulpy (drupes) and turn purple-black at maturity. In Mt. Kenya region the fruits are sold in markets for human consumption at a small scale, nevertheless contributing to the nutrition of the local population.
3.3 Reproductive biology of V. keniensis and V. fischeri
In the first attempts to execute controlled pollination from January to March 1996, most of the flowers aborted. That could be attributed to unusually dry weather conditions during that year. The experiments were repeated in 1997 and 1998. Prior to the pollination experiments to discern the species mating systems, the stage for optimum pollen viability and stigma receptivity were assessed in each of the Vitex populations. This is because pollen viability and stigma receptivity affect the success of pollination and the subsequent fertilisation process and seeding. The studies were also carried out to detect temporal separation of the sexual stages, if any.
3.3.1 Viability assessment of V. keniensis and V. fischeri pollen grains
Materials and methods The viability of pollen grains of the species was tested at different stages of flower development from the onset of anthesis at the beginning of flowering season. Two methods of analysis wereapplie d onth epolle nviabilit y assessment; staining by aniline blue and by tetrazolium salt (Dafni, 1992). The tests on pollen viability were crucial inensurin g that thepolle ngrain s used inartificia l pollination experiments wereviable .
Five trees were marked ineac h of the four Vitex populations and 20flower s of each sampled tree tagged at theonse t ofblooming . Pollen grains were collected using a fine brush and their viability was tested daily by staining, starting from just before anthesis for up to 20 days when the flowers wilted and the corolla and stamens are shed. The viability of pollen grains were tested by the two staining methods as a precaution against over- or under-estimation.
46 Aniline blue test on the viability of Vitex pollen grains 1. Samples ofpolle ngrain s takena tdifferen t stageso fflowe r development werepu t on a glass slide and a drop of 1% anilin e blue was added. 2. The pollen sample was covered with a glass cover slip. 3. The dye solution was replaced with water after 5 minutes of staining. 4. The pollen grains were put under a microscope at x40, the stained pollen counted, and the % of the stained pollen was considered as a measure of its viability.
Tetrazolium tests on viability of Vitex pollen grains. This was done by the method of Heslop-Harrison et al. (1984) and Stanley & Linkens (1974). 1. Samples of pollen grains werepu t ina dro p of 0.5% , 2,3,5-triphenyl tetrazolium chloride in 35% sucrose solution and covered immediately to exclude oxygen, which can inhibit dye reduction. 2. The preparation was kept at 50°C in the dark for 2 hours. 3. The red-coloured pollen grains whichwer e atth e central area of the sample were counted under a microscope. 4. The proportion of the stained pollen was determined and converted into %, and taken as a measure of the pollen viability.
Table 3.1. Analysis of Variance on Pollen Viability of Vitex populations of Mt. Kenya, Nyambene hills, Mt. Elgon and L. Victoria (* denotes significance at p < 5% and ns non-signi ficance).
Source of Variation DF MS F
Staining Methods 1 5916.72 132.21 * Vitex populations 3 344.34 7.69 * Residual 3 44.75
Table 3.2. Means of the viability of pollen grains of the Vitex populations from Nyambene hills, Mt. Kenya, Mt. Elgon and L. Victoria (means denoted by the same letter are not significantly different atp < 5% significance level according to Tukey Test).
Vitex Population Mean staining % of pollen grains
Aniline blue Tetrazolium salt
Mt. Kenya 28.810ab 18.381ab Nyambene hills 24.476a 14.381a Mt. Elgon 32.857b 18.286ab Lake Victoria 31.619b 19.238b
47 • Mt. Kenya .Q > • Nyambene Mt. Elgon O Q. C L. Victoria ra
4 6 8 10 12 14 16 18 20 Numbero fday sfro manthesi s Figure 3.3. Trends in the mean pollen viability of V. keniensis and V. fischeri populations.
Results and discussions The pollen staining methods differed significantly (P < 0.05) from each other. The pollen stainability by tetrazolium salt was lower than that of aniline blue. However, the difference was relatively constant in most samples. The observations indicate the difference in the sensitivity of the two methods in assessing pollen grain viability.
The two methods have their strengths and weaknesses; the advantages of using of anilineblu e aretha t it iseas y tohandl e and it allows large samples tob e analyzed, but insom e cases itma y showlo w correlation to therea l germination level invivo; theus e of tetrazolium tests is rapid and correlates fairly well with pollen viability and is also suitable for analysing large samples, but the set-back is that desiccated pollen grains in some instances may give false-positive results (Dafni, 1992). The use of the two pollenviabilit y testingtechnique swa sa cautionar y measureagains tdrawin g inaccurate conclusions. The average of the two methods was therefore considered to reflect the estimates of the pollen viability level of pollen grains of Vitex. The four Vitex populations differed significantly (p < 0.05) in their pollen viability (Table 3.1).
Generally, from the mean pollen viability test results (Table 3.2) the pattern of variation was somewhat consistent with the ecological range of the Vitex populations. Nyambene hillsdi d notdiffe r (P < 0.05) significantly from Mt. Kenya. The samewa s observed between Mt. Elgon and Lake Victoria populations. Mt. Elgon Vitex had the highest pollen grain viability and they attained maturity much faster than the other populations (Figure 3.3). The slow pollen maturation and lower pollenviabilit y inth e Nyambene hills and Mt. Kenya could be attributed to the mild temperatures and wet conditions in the habitats of the two Vitex populations. Pollen grains from Lake
48 Victoria had a relatively high viability level but the loss in viability was quite rapid after the peak. This is probably because of the high temperatures and relatively high humidity from the Lake. The variation of the level of pollen viability and maturation between the different populations of Vitex is a possible indication of the role of environmental conditions. Similar observations have also been made by Johri &Vasi l (1961), Stanley &Linsken s (1974)an d Stephensone t al. (1992)o nth einfluenc e of the prevailing temperature and humidity on the competence of pollen.
Mt. Kenya and Nyambene hills Vitexreac h optimum pollen maturity 9 days after anthesis while in Lake Victoria and Mt. Elgon it takes place 6 days after anthesis. In all cases, pollen grains lost their viability in about 17 days. Therefore, for higher vigour of Vitexpolle n grains, it is necessary to collect them 9 days after anthesis in Mt. Kenya and Nyambene hills but 6 days after anthesis for Lake Victoria and Mt. Elgon. Further research is needed to determine the storage life of the pollen grains to enhance the species' ex-situconservatio n and breeding programmes.
3.3.2 Evaluation of stigma receptivity of V. keniensis and V. Jischeri
Stigma receptivity is a crucial stage in the maturation of the flower which may greatly influence the rate of self-pollination and pollination success at different stages in the flower life cycle (Galen et al., 1987). Therefore any success in breeding experiments or artificial pollination procedure should be accompanied by tests on timing and duration of stigma receptivity. Stigma receptivity can be determined experimentally by examining morphological changes such as colour and presence of exudate, determination of pollen germination or tube growth or fruit set after pollination at different times relative to the flower opening and by staining or testing for esterase presence (Dafni, 1992).
The assessment of stigma receptivity in Vitexwa s carried out in-vivob y artificial pollination of flowers at different stages of development. Flowers of sampled trees were emasculated, hand-pollinated at different stages of development (starting just before anthesis to wilting stage) and bagged to isolate the flowers from foreign pollen grains. That was expected todetermin e the optimum flower age in the different Vitex populations for the subsequent artificial-pollination experiments.
Materials and methods 1. Ten trees were randomly sampled in each of the four Vitexpopulation s at the beginning of flowering season. 2. 20 flowers were marked and tagged on each sampled tree. 3. The flowers were thenemasculate d and hand-pollinated ata n interval of oneda y (starting from onset of anthesis) with viability tested samples of pollen grains. 4. The number of fruits resulting from each day's treatment was considered an estimate of stigma receptivity.
49 Table 3.3. Analysis of variance on stigma receptivity of V. keniensisan d V. fischeri (* denotes significance at 5% level).
Source of variation DF MS F
Population 3 42.47 6.02* Tree 9 2.68 0.38* Residual 27 7.05
Table 3.4. Means of the number of fruits formed by the V. keniensisan d V.fischeri population s following hand-pollination.
Vitex Population Mean number of fruits formed
Mt. Kenya 4.181b Nyambene hills 4.490b Mt. Elgon 3.814a Lake Victoria 3.452a
Results and discussions Thenumbe r of fruits formed wasassume dt oindicat e theleve lo f stigma receptivity. Thefou r populationsdiffere d (P < 0.05) significantly from oneanothe r inthei r stigma receptivity (Table 3.3). There was also a significant difference between trees in each of the populations.The observation could be attributed to the genetic make-up of the individual trees or micro-environmental variation.
The stigma receptivity level was higher in the flowers of Nyambene hills and Mt. Kenya populations and lower in Mt. Elgon and LakeVictori a populations (Table 3.4). This iscontrar y to their pollen viability levels. However, the stigmas of Lake Victoria and Mt. Elgon Vitexpopulation s became receptive earlier, reaching the peak 6 days after anthesis, while the Nyambene hills and Mt. Kenya populations reach maximum stigma receptivity 10day s after anthesis (Figure 3.4). The rapid stigma development and decline in receptivity after the peak in the Lake Victoria and Mt. Elgon populations could beattribute d tohighe r temperatures inth e two regions. Thedelaye d stigma maturation inNyamben e hills and Mt. Kenya Vitex populations may bea result of the lower temperatures and high humidity. The stigma receptivity in Lake Victoria and Mt. Elgon Vitex is critical, the graph assuming a conical shape which declines rapidly after reaching the peak. The Mt. Kenya and Nyambene hills Vitex populations were more prolific, producing large number of fruits and Lake Victoria had the least, thus exhibiting the lowest reproductive capacity. This suggests that in Vitex, stigma receptivity has a major role in successful pollination and seed production. Therefore, despite exhibiting slow pollen maturation and low pollen viability, Mt. Kenya and Nyambene hills Vitex produced the highest number of fruits. The observed significant differences within the Vitex populations in stigma receptivity was possibly caused by the differences in the ecological zones by the different populations of the species.
50 I
'5 •Mt . Kenya •Nyamben e -Mt .Elgo n n E •L .Victori a =J C C (0 (1)
4 6 8 10 12 14 16 18 20 Number of days from flower anthesis Figure 3.4. Trends in stigma receptivity in V.keniensis an d V.fischeri populations .
3.3.3 Pollinationmechanism san dself-compatibilit y inV. keniensisan dV. fischeri
Materials and methods Pollination experiments were carried out to investigate mating parameters of the Vitex populations. The methods of Dayanandan et al. (1990) was applied, but with a few modifications. Iuse d numbered weather-resistant tags and exclusion bags madeo f perforated plastic material. Ten Vitex trees were sampled per population and 20 flowers were tagged for each treatment on each tree. The flowers were marked at anthesis stage and controlled pollination carried out 5t o7 day slate r for Mt. Elgonan d Lake Victoria populations but after 8t o 10day s in Mt. Kenya and Nyambene hills as recommended in the previous pollen viability and stigma receptivity assessments. A large isolation distance of at least 1 km was between the randomly sampled trees was maintained toavoi d biparental inbreeding associated withoutcrossin g ofclosel y spaced individuals and to ensure coverage of the species range.
Hand-pollination treatments on of the Vitexflower s 1. NEO Flowers non-emasculated and left open Theflower s wereunmanipulate dan dacte d asth econtro l for comparison withth eothe r treatments to detect the effects of emasculation and the species' reproductive capacity under natural conditions. 2. NEB Flowers non-emasculated and bagged The flowers were unmanipulated but bagged to exclude foreign pollen sources. Fruit set in this treatment is an indication of apomixis or possibility of spontaneous self- pollination without pollinator. 3. EB Flowers emasculated and bagged The anthers were removed by fine forceps. It is the best way to test for autonomous
51 apomixis (Richards, 1986). If seeds are produced without pollen, then pollen is not necessary and the seeds are apomictic. If no seeds are formed then either pollen grains are necessary or the flowers were sotraumatize d by the treatment that it aborted. The latter can be ruled out by comparison of seed set of the treatment with that of emasculated and crossed, and emasculated and selfed. 4. NESB Flowers non-emasculated, selfed and bagged The flowers were unmanipulated but selfed and bagged. Comparison of fruit set with the other treatments gives an indication of the effects of emasculation on pollination. 5. ESB Flowers emasculated, selfed and bagged The anthers were plucked out using fine forceps, the flowers selfed and bagged. The result from this treatment is to show the degree of self-compatibility or pseudogamy in the species. If seeds are set, then the species is either self-compatible or pseudoga- mousapomicti c (Nybom, 1989;Campbel l &Dickinson , 1990).However , lack of seed set is more difficult to interpret. It could also mean that thepollinatio n technique was ineffective or the flowers were damaged by the treatment (emasculation), or that the flowers wereself-incompatible . Thesepossibilitie s canb eteste db ycomparin g thesee d set from naturally open pollinated flowers (1) and emasculated and crossed treatment (7). 6. NECB Flowers non-emasculated, crossed and bagged The flowers were unmanipulated, crossed and bagged to assess the effect of emasculation on fruit set, for comparison with other treatments. 7. ECB Flowers emasculated, crossed and bagged The flowers had their anthers removed, crossed with a mixture of pollen from the different Vitex trees and bagged. The treatment was carried out todetec t the degree of out-crossing in the species within the populations. 8. EO Flowers emasculated and left open. The anthers were removed and the flowers left exposed to open pollination to assess if wind-pollination or cross-pollination by insects can occur.
Results and discussions In the controlled pollination experiment, the different flower treatments varied significantly at p < 0.05. But there was no significant (p < 0.05) difference within and even between the Vitex populations (Table 3.5). This is a possible indication that the studied Vitex populations had a similar mode of pollination besides sharing similar floral morphology. The number of fruits formed varied from one treatment to another (Table 3.6).
The species produced fruits by both self-an d cross-pollination. The summary of the assessments on the degree of each of the two modeso f pollination are presented inth e next section (Table 3.7). The mixed mating system in the species is evident from the formation of fruits by flowers that were emasculated, selfed and bagged produced fruits as well as the ones that were emasculated, crossed and bagged. The amount of fruits formed by flowers which were emasculated and bagged was negligible; an
52 indication of the need for pollen for fertilisation and possibly the absence of apomixis in the species. The few seeds formed in that treatment could have been from contamination of the flowers before the treatment. The tree species showed high seed production under the natural open pollination conditions, a characteristic commonly associated with self-compatible and self-pollinated plant species (Dafni, 1992). However, it is worth noting that bagging treatments do increase the temperature, thus can negatively affecting pollination of flowers.
Table 3.5. Analysis of variance of controlled pollination on V. keniensis andV. fischeri ( *denote s significance atp < 5% level and ns non-significance).
Source of Variation DF MS F
Treatment 7 726.81 166.77* Population 3 4.18 0.96ns Pop*Treat 21 1.72 0.39ns Pop*Tree 27 3.93 0.90ns Residual 261 4.36
Table 3.6. Means of the number of fruits formed out of 20 flow ers (per tree in each population) following different the hand- pollination treatments on V. keniensis and V. fischeri (Means denoted by the same letter are not significantly different at 5% significance level according to Tukey's Test).
Flower Treatment Mean no. of fruits formed after hand pollination of 20 flowers per treatment.
NEO (Control) 14.425e NEB 3.850cd EB 0.050a NESB 4.500d ESB 4.125cd NECB 4.100cd ECB 2.950bc EO 2.100b
The success in pollination and fruit formation in the non-emasculated treatments in the four Vitex populations showed a similar trend and decreased in the order of open, selfed, crossed and bagged treatments (Figure 3.5). The fruit set in non-emasculated and bagged treatments, suggested that pollination could occur in the species' flowers passively, without intervention of pollinators possibly by gravity or direct contact between the stigma and anther. In the flowers, the anthers are above the stigma so there isa possibilit y of thepolle nbein g dislodged by gravity at maturity and deposited
53 B Mt. Kenya a Nyambene jS Mt. Elgon I B L. Victoria
NEO NEB EB NESB ESB NECB ECB EO Flower pollination treatments Figure 3.5. Effects of the different hand-pollination treatments on the production of fruits in the V. keniensisan d V.fischeri populations . on the stigma. Fruit-set from non-emasculated treatments was higher than the emasculated ones, indicating a possible negative influence of flower emasculation on fruiting through mechanical injury. Since most insect pollinators visit flowers for pollen grains, they tend to avoid emasculated flowers (Cruden et al., 1989; Young & Stanton, 1990; Donnelly, 1998). This can also lead toth e low number of fruits among the flowers that were emasculated and left open. In some cases the stigma and style may function abnormally following emasculation thus leading to formation of lower numbers of fruits (Guo et al., 1990).
3.3.4 Estimates of the mating parameters of V. keniensis and V. fischeri populations
The data from the previous hand-pollination experiments were used for estimating the species' matingparameters , sucha sSelf-incompatibilit y (ISI), Selfing rates (S)an d Relative Reproductive Success (RRS).
Self-incompatibility inth e V.keniensis andV. fischeri population s was estimated by the method of Zapata & Arroyo (1978). In this method, self-incompatibility is estimated as proportion of fruits formed by cross-pollination to those from self- pollination. According to the method, the values of the self-incompatibility reflect the following possibilities: > 1 = self-compatible > 0.2 < 1 = partially self-incompatible < 0.2 = mostly self-incompatible 0 = completely self-incompatible
54 Based on the categorisation by Zapta &Arroy o (1978), the four V.keniensis and V.fischeri population s arepartiall y self-incompatible (Table 3.7). However, innatur e selfing rates may becontrolle d byothe r adaptive features such asdichogamy . If self- compatibility rates are genetically controlled as observed by Dafni (1992), then the similar levels of partial self-incompatibility in the different Vitexpopulation s could imply their taxonomic or genetic similarity.
Table 3.7. Theleve l of self-incompatibility inth e Vitex populations.
Population No. selfed fruits No. crossed fruits Self-incompatibility formed formed levels
Mt. Kenya 34 27 0.8 Nyambene hills 39 29 0.7 Mt. Elgon 52 28 0.5 Lake Victoria 40 33 0.8
According to Richards (1986) most polyploids tend to suffer from inability to reproduce sexually andestablish , unless they areself-fertile . That mayb eon eo fth e explanations for the observed partial self-incompatibility in Vitex. Self-compatibility has proved more successful inpolyploid s than indiploids , perhaps duet oth egreate r capacity ofpolyploid s for storing variability, and releasing itmor e slowly, when selfed (Richards, 1986). Thisphenomeno n makespolyploid s more capableo ftoleratin g more selfing than indiploids .
However, in spite of self-compatibility being a desirable asset in a number improvement breeding strategies, in several instances selfing is associated with inbreeding depression. Therefore, thenex tsectio npresent sth eevaluatio no fth evigou r of the seeds (offspring) from self- and cross-pollination by assessing and comparing their germination levels.
3.3.5 Comparison of the germination vigour of seeds from cross- and self- pollination of V. keniensis andV. fischeri
The experiment was carried outa smeasur eo fvigou r ofoffsprin g resulting fromth e different pollination treatments.
Materials and methods 1. Seeds from open-, cross- and self-pollination were processed, labelled and kept separate per population. 2. Theseed s were sown invermiculit ei n4 replicate s of50 , kept moist by watering and put ina n incubator seta t 25°C . 3. Their germination wasrecorde d daily until nomor e seeds were germinating. 4. Theresult s were summarised andexpresse d in%.
55 c o CO c
D) •a
Mt. Kenya Nyambene Mt. Elgon L.Victori a Vitex population Figure3.6 . Germination vigour ofV. keniensis and V.fischeri seed s from open-, self- and cross-pollination.
Results and discussions The average germination ofth e species' seeds isnormall y ranging from 25 to 50% (Rhode, 1986) andi tha sbee n attributed todormanc y imposed byth ehar d nuto n the seed. Therefore, the low germination recorded is normal for the tree species. However, in general seeds from cross-pollination had higher germination than those from open-an dself-pollinatio n excepti nNyamben ehill s (Figure 3.6). Contrary to my expectation seeds from open pollination showed lower mean germination than those from self-pollination. Theseed s from cross-pollination exhibited slightly higher mean germination than theothe r twomode s ofpollination . However, thedifferenc e insee d germination amongth e Vitexpopulation s wasmarginal . Thedifferen t Vitexpopulation s did not differ much in the degree of loss in seed germination capacity among their selfed offsprings possibly indicating alo wdegre e of inbreeding depression. Themil d inbreeding depression could bedu et oth ehig hpolyploid y level ofth e species, 2n=96. The phenomenon hasals o been observed byRichard s (1986). Therefore, it seemsth e Vitextree s can also be selfed without the risks of loss in seed set and germination capacity. According to Curtis (1999), species exhibiting high, persistent inbreeding depression is unlikely to evolve towards increased self-fertility. Therefore, the self- compatibility in Vitexha spossibl y been maintained by thespecies ' ability to tolerate or buffer inbreeding depression.
Further research isnecessar y toasses s theeffect s ofselfin g onothe r desirable traits of the species. This isbecaus e although inbreeding depression was notdetecte d in the study insee d setan d germination, iti sessentia l tosampl e indices offitnes s throughout the life cycle of the progenies. Inbreeding depression may bedetecte d ata later stage (e.g. Belaoussoff & Shore, 1995). In some cases inbreeding may not be detectable unless theplant s are subjected to environmental stress (Eckert &Barrett , 1994).
56 Table 3.8. The degree of selfing rates (S) inV. keniensis andV. fischeri populations . The calculations were done according to Charlesworth & Charlesworth (1987).
Population Fruits from Fruits from Fruits from Estimates of cross- open self- selfing rates pollination pollination pollination
Mt. Kenya 27.00 143.00 34.00 16.57 Nyambene hills 29.00 140.00 39.00 11.10 Mt. Elgon 29.00 149.00 52.00 05.22 Lake Victoria 33.00 145.00 40.00 16.00
Estimates of selfing rates (S) in V. keniensis and V. fischeri were calculated according toth emetho db y Charlesworth &Charleswort h (1987). Self-pollination rate is a criterion used to assess the fate of pollen on the stigma of the same flower or plant. A plant species may be self-compatible but exhibit low selfing rates due to timingevent s inth eflowers , itsstructur e andpollinator' sbehaviour . Therefore, selfing rates need to be evaluated besides the degree of self-compatibility.
The number of seeds from the different modes of pollination were compared according to Charlesworth & Charlesworth (1987) in estimating selfing rates in the various Vitexpopulations . Selfing rate is an important criterion in evaluating the fate of pollen on the stigma of the same flower of a plant. The method makes it possible to estimate the rate of selfing of a species in natural conditions (Dafni, 1992).
Vitexi n Mt. Elgon showed the lowest selfing rates while those of Mt. Kenya and Lake Victoria the highest (Table 3.8). That implied that populations with high self- compatibility may not necessarily havehig h selfing rates. It supported the observation made by Dafni (1992) that aplan t may be highly self-compatible but has low chances of self-pollination. So there may be a range of factors responsible for the low selfing rates observed in Mt. Elgon populations such as climatic conditions.
Estimates of Relative Reproductive Success (RRS) in the Vitexpopulation s were done according to Stanton & Preston (1988). The estimates of relative reproductive success were as follows; Mt. Kenya had 0.179, Nyambene hills 0.175, Mt. Elgon 0,181an d Lake Victoria 0.186. Thesevalue s arerelativel y lowbecaus eou t of the four ovules in the ovary of Vitex only one of them gets fertilised to form viable seed. The reason for this is not yet established but it could be a result of infra-fruit competition arising from resource limitation, thus influencing the final number of seeds per fruit and even the spacing of seeds in the fruit (Stanton & Preston, 1988). According to Stephenson et al. (1992) such selective abortion does improve the quality of the few seeds produced. The average relative reproductive success in Vitex is about 0.183. In nature, the species flowers and fruits profusely, that could be in response to the need to compensate for the low relative reproductive success.
57 3.3.6 Reproductive compatibility between the V. keniensis and V. fischeri populations and the vigour of the resultant seeds
The four V.keniensis an dV. fischeri populations were crossed and the germination of the resultant seeds tested. The experiment was designed to determine the reproductive compatibility between the different Vitex populations and the vigour (germination) of the resultant seeds. The results were compared with the number of fruits formed from cross- and self-pollination. The study aimed at finding out the possibilities of outcrossing the different Vitexpopulation s for possible improvement breeding work inth e future andt odetec t ifther e isreproductiv e barrier between them.
Materials and methods Ten trees in each of the four populations were selected and 10flower s on each of them used per treatment. The flowers were emasculated and artificially pollinated by viability tested pollen from other populations (crossed) and bagged to avoid contamination from foreign pollen grains. The controls were marked and left to open pollination. The fruit development was monitored and harvested at maturity.
All the fruits obtained from the different treatments were extracted, the seeds processed, labelled and sown in vermiculite in four replicates. This is because the number of seeds obtained from thehand-pollinatio n was not enough for four replicates of at least 25 seeds (ISTA, 1993). The seeds were sown in vermiculite and put in a germinationcabine t seta t2 5° Can dmoistene d withwater . Germination was monitored and recorded daily until nomor e seeds were germinating and themea n germination % were calculated for each seed lot (Table 3.9).
Table 3.9. Number of fruits formed from open-pollination (*) and cross-pollination of the V. keniensis and V.fischeri populations and their respective seed germination percentage. A: Number of fruits formed; B: Seed Germination %.
Female Mt. Kenya Nyambene Mt. Elgon L. Victoria
Male A B A B A B A B
Mt. Kenya 72* 35%* 55 32% 48 14% 32 20% Nyambene 53 26% 75* 39%* 50 19% 44 12% Mt.Elgon 43 12% 34 11% 70* 24%* 56 17% L.Victoria 26 9% 28 8% 39 13% 73* 47%*
Results and discussions The results show that it ispossibl e tocros s thedifferen t Vitexpopulation s and obtain viable seeds. More fruits and higher seed germination rates were recorded from the controls (open-pollination) (Table 3.9). The higher number of fruits from the control was probably because they were not subjected tomechanica l damage by emasculation. According to Stuessy (1990) themor e robust and fertile thehybrids , the closer will be
58 their genetic relationship. The observed germination of the out-crossed seeds is therefore a possible indication of close taxonomic or genetic relationship between the different Vitex populations. However, the long time scale involved in raising the tree species could not allow further assessment of offspring survival, performance and fertility upon maturation. However, it is necessary to monitor the performance of the progenies throughout a complete life cycle. That is because sometimes inbreeding depression may set in at a later stage of development or when they are faced with a stressful environment.
3.4 Conclusion
The similarity in the floral morphology between the Vitex populations could be a reflection of their common ancestry and lack of taxonomic distinction between them. They also exhibited similar sequence in their phenology although the timing and duration differed according to the ecological zones. That is a possible indication that the phenological pattern of the Vitex is influenced by the environmental conditions.
The differences inpolle n viability and stigma receptive among the populations also tended tomatc hth eecologica l andgeographica ldistributio npatterns . Thephenomeno n could be attributed toth e influence of temperature and moisture on thepolle n viability and longevity. The pollen viability is normally sensitive to environmental conditions especially temperature and moisture (Vogler et al., 1999). According to Vogler et al. (1999) also found thatho t environments led tohig hpolle nviability . Therefore thehig h pollen viability inLak e Victoria and Mt. Elgon Vitex could bea resul t of the relatively high temperatures in these regions. This is an indication that pollen viability inVitex is affected by the prevailing environmental conditions.
The tree species has a mixed mating system. The partial self-incompatibility observed in the Vitex populations is possibly an adaptation to maintain fecundity even whenpollinato r availability isunpredictable . Pollinator-limited seedproductio ni s often presumed to favour evolution of self-compatible from self-incompatible ancestors (Wyatt, 1983, 1984; Dafni & Bernhardt, 1990; Weisler & Snow, 1992). SinceVitex is capable of producing seeds both by selfing and crossing. The level of selfing and crossing would depend on other factors such aspopulatio n density and the availability of pollinator. Therefore, reduction in its population size and density is likely to promote selfing and inbreeding. Over time this can lead to reduction in genetic variation, inbreeding depression and reduced adaptability of its populations to environmental conditions.
Biparental inbreeding is also common where seed dispersal is limited. Vitex seeds are normally dispersed by monkeys, hornbill birds and to a limited extent humans. Continued exploitation of the forests might lead to destruction of the habitat and decline in the populations of seed dispersal agents. Over time, biparental inbreeding also induces the evolution of selfing in previously outcrossing species (Lloyd, 1979;
59 Uyenoyama, 1986). Outcrossing mating systems are common in most tropical tree species (Loveless, 1992; Bawa, 1992). Thepartia l self-compatibility in Vitex could be arising from biparental breeding as a result of the decline in its population, seed dispersal agents and pollinators.
Selfing canevolv e repeatedly inout-crossin g taxa, andtheor y predictstha t increases inth eleve l of self-fertilisation occur inconcer t withchange s inreproductiv e allocation and the magnitude of inbreeding depression (Parker et al., 1995). The results from fitness inconsequenc e ofselfin g andcrossin g indicated somelo wdegre eo fvigou r loss in terms of seed production and germination levels. The apparent mild consequences of selfing, that is inbreeding depression in Vitex,coul d be attributed to its polyploidy and high heterozygosity. This concurs with the observation made by Lande & Schemske (1985) that diploids exhibit higher inbreeding depression than tetraploids (Richards, 1986). The phenomenon can enable Vitex to withstand the negative effects of inbreeding for some generations.
The possibility to cross the different Vitexpopulation s is a reflection of genetic compatibility in a particular evolutionary line (Stuessy, 1985). This implies that the different Vitexpopulation s have not yet undergone reproductive isolation. The high degree of reproductive compatibility between the Vitex populations is therefore a further indication of the close similarity between them inadditio n tothei r morphologi cal traits. The inter-population reproductive compatibility means that desirable traits can easily be transferred from one population to another through a breeding programme.
60 4 The genetic structure of V. keniensis and V.fischeri populations
4.1 Introduction
In developing sampling strategies for conservation efforts or genetic improvement of tree species, it is useful to take into account overall geographic patterns of genetic differentiation. The geographical distribution of genotypes constitutes the spatial structure of apopulatio n revealing pockets of diversity (Boshier et al., 1995; Kuser et al. 1997). The genetic structure is influenced by and influences all aspects of species' population genetics such as mating system, seed and pollen dispersal, and natural selection (Epperson, 1992). Theknowledg e of thestructur e of geneticvariatio n isvita l in understanding the population genetics of a species as well as for the practical application of the knowledge in the sustained management of production forests (Finegan, 1992). It can also help us to understand the historical processes leading to the genetic diversity of a species (Sheely & Meagher, 1996). The value of data on geneticvariatio n for conservationha sgaine dincreasin g recognition (Falk& Holsinger , 1991). Large scale deforestation can lead to reduction in effective population size and genetic isolation of fragmented populations. Maintenance of the genetic diversity is considered crucial for long-term survival and evolutionary response of populations to changes inth eenvironmen t (Hueneke, 1991)thu spreservin g itsevolutionar y potential. Taxonomy also plays acritica l role in conservation since only formally named entities can be afforded legal protection (Avise, 1994). Taxonomic uniqueness has also been promoted as a factor leading to priority conservation status. Therefore, understanding genetic relationships of taxa may help define taxonomic entities and determine their conservation priorities (Stewart et al., 1996).
A common consequence of human interventions in plant populations is mostly fragmentation during exploitation, or clearing for other land uses. Depending on the scale and duration, these interventions could reduce genetic diversity. Inrecen t years, information pertaining topattern s of genetic variation in forest tree taxa has increased (Hamrick & Godt, 1989; Hamrick et al., 1992). The upsurge can be attributed to several factors, among others, to the growing awareness of the role of forestry in biodiversity conservation (Williams, 1991), the need to evaluate genetic diversity before andafte r management interventions (e.g. Bawa& Krugman , 1991; Salvonainen & Karkkainen, 1992), the desire to understand genomic organisation for tree improvement programmes (Neale &Williams , 1991), and the availability of new and refined techniques for gathering this information (Cheliak &Rogers , 1988). Although there has always been the need to define patterns of forest tree populations for better exploitation and management, it is the current refined assay techniques that have revolutionised the investigations, reducing the time required for analyses and decision making.
61 The use of markers in plant population biology is increasing and the knowledge about their applications, choices of techniques and dataanalysi s for molecular markers has become an important issue (Vekemans & Jacquemart, 1997). Molecular markers such as allozyme loci have been used in a wide array of applications in the field of plant biology such as population genetic structure, genetic diversity of natural populations and breeding systems. In the context of conservation management, molecular markers have been used gather genetic information on endangered plant species to aid sampling for ex-situ conservation (Schoen & Brown, 1995). Other application of molecular markers include phylogenetic analysis (Swofford et al. 1996), ecological genetics (Azzouzi et al., 1997), detection of hybridisation (Heinze, 1997) and cultivar identification (Hillis, 1996). The discovery that genes code for proteins, combined with the development of thetechnique s of protein electrophoresis, provided means of estimating genetic variation in natural populations (Crawford, 1990). These analyses have investigated various species, among them some forest tree species (e.g. Hamricket al., 1992; Moran, 1992; Zimniak-Prybylska, 1995).
A number of other techniques have been developed to evaluate the genetic characteristics of plant populations (Crawford, 1990; Murphy et al., 1990; Neale & Williams, 1991). The choice of the technique depends on the aims of the study, the sensitivity and convenience of the technique, and availability of resources. Currently, these evaluations are implemented using methodologies including DNA (deoxyribonucleic acid) sequencing, gene mapping, restriction fragment length polymorphisms (RFLPs), randomly amplified polymorphic DNA (RAPDs), and characterisation of seed storage proteins and specific proteins, including isozymes.
DNA sequencing is the most informative of all the molecular techniques, as it determines all the genetic variation (Crawford, 1990). However, the cost of DNA sequencing has limitations to its wider application. The use of DNA fragments, for example, RFLPs and RAPDs, have been developed as methods of intermediate cost (Neale &Williams , 1991; Krutovskii &Wagner , 1992). However, their costs arehig h compared to the isozymes, the use of radioisotopes (RFLPs), the limited number of probes (RFLPs), the long processing time (RFLPs) and the problems associated with dominant bands hence difficulty of scoring heterozygotes (RAPDs) make them less popular than isozymes (Neale & Williams, 1991). The isozyme technique is at the moment, therefore, one of the most cost-effective methodologies presently available for many purposes, and can be widely adopted for genetic analyses.
The distribution of genetic diversity of plant populations have been widely documented by means of allozyme markers for the last 20 years (Mahy & Neve, 1997). The studies ontropica l forest tree species indicatetha tthe y maintainhig h levels of genetic diversity within populations, and are far less geographically differentiated thanherbaceou splant s (Hamrick etal. , 1992). Theultimat e goalo fthes e investigations was to characterise Vitexpopulation s in terms of their taxonomic status and genetic structure. Analyses of isozyme patterns of different taxa have proved to be of great value in establishing patterns of genetic variation, investigating evolutionary genetics,
62 and in the verification of crossings, hybridisation, introgression, ploidy and paternity (Adams, 1983; Murphy et al., 1990). Some of the characteristics that make isozyme markers appropriate for genetic studies were summarised by Brown & Allard (1970) and Brown et al. (1989).
Despite having been documented by many workers, gel electrophoresis has no universally ideal single system of assay, even within one species. A set of optimum conditions have to be established empirically, depending on the organism, tissue, enzyme system and, at times, the locus (Wendel & Weeden, 1989). However, what ever the source of variation is, polymorphism is useful as it reflects genetic control.
The principle of electrophoresis (isozymes) is based on the nature of amino acids. Amino acids have positive or negative charges but some are neutral, this leads to the differences in their mobility in an electric field. The variation in patterns is assumed to reflect amino acid substitution in an enzyme which leads to the difference in electrophoretic mobility on the supporting medium (Freifelder, 1976). Various supporting media such aspaper , cellulose acetate, agarose, starch and polyaer ylamid e gels have been used (Feret &Bergmann , 1976; Freifelder, 1976; Wendel &Weeden , 1989; Murphy et al., 1990). Starch and polyaer ylamid e gels are widely employed (Freifelder, 1976). The electrophoretic data are gathered as isozymes and allozymes (Murphy et al., 1990), which are expressed as bands of different mobilities after staining. The bands reflect thealleli c variation inth eorganism , theyca nb e scored and interpreted to express genetic diversity (Gottlieb, 1981). To reduce loss of enzyme activities and protein denaturation, the tissue should be homogenized in a short time in an appropriate extraction buffer.
In this study, trees were sampled in the four V. keniensisan d V. fischeri popula tions,th eoptimu melectrophoreti c assaycondition swer edetermine db y standardization of tissue homogenates, thebuffe r systems andstainin g requirements optimized withth e aim of achieving comparable isozyme resolutions. Theresultan t isozymeprofile s were used to elucidate the nature of polyploidy, segregation pattern, taxonomic status and genetic structure of the Vitex populations.
4.2 Analyses of the genetic structure of V. keniensisan d V. fischeri populations
4.2.1 Sampling and isozyme analysis methods
The natural geographically isolated populations of Vitex in East Africa were identified andmappe d (Figure2.3) . Ahundre d individual treeswer erandoml y sampled and marked in each of the Vitex populations (Mt. Kenya, Nyambene hills, Mt. Elgon and Lake Victoria). A minimum distance of 250 m was maintained between the sampled trees with the aim of reducing the chance of assembling closely related individuals and to maximise the coverage or representation of the species' natural
63 population range. The appropriate sample size is still a debatable question in plant population analysis (Crawford, 1990; Bawa; 1992), but it is clear that the larger the sample size, the more accurate the estimated parameters.
Fifteen enzyme systems (Appendix 4.1) were first screened on Vitex. Then the optimum electrophoretic assay conditions of the best 8 were refined, which included standardisation of tissue homogenates, optimisation of buffer systems and staining requirements according to Wendel and Weeden (1989), with the aim of achieving comparable isozyme resolutions. Different tissues of Vitex such as seeds, seedlings (cotyledons and embryos), fresh leaves, roots and barks were tried out.
Homogenisation of samples A complex extraction buffer of Tris-HCl (Wendel &Weeden , 1989) wasuse d with some modification. The roles of buffer additives are explained by Kephart (1990). To minimise the variation arising from physiological and ontogenetic conditions, only materials atth e samedevelopmenta l stage were homogenised anduse d inth e analyses. An extraction buffer (0.5 ml) was added to each sample and extraction performed in labelledmicrotube s overice . Alternatively, microtubeswit hbuffe r werefroze n at-4° C in a freezer prior to the addition of samples. The frozen buffer cooled the tissues and also helped in crushing before thawing. The plant materials (seeds, seedlings, leaves, bark and roots) were mechanically crushed with a tapered glass rod in the microtubes because grinding with chilled mortar and pestle was time-consuming and prone to contamination. Consequently, the use of a power homogeniser was inappropriate because of warming up. The glass rod was washed and rinsed with absorbent paper between samples to avoid contamination. The samples were loaded onto the gels and electrophoresis machine immediately. Delays or storage of the samples led to low enzymeactivit y andcause dpoo r resolution. Toavoi d delays onlyhomogenate s enough for one day's work (i.e. 20 samples for 8 enzyme systems) were prepared. The homogenates were centrifuged at 1100r.p.m . for 3minute s at room temperatures (20- 25°C) . Otherwise longer centrifuging time led complete loss of activity of some enzymes such as ADH and ME and this could be attributed to warming of the homogenates in the process. The supernatant was decanted and a 10 /xl used to load the gel. The 20 samples were loaded into the 20 wells of each gel which had been filled with the electrode (tank) buffer. Samples being denser than the buffer, they settled at the bottom of the wells.
The apparatus was then assembled and each tank run for 4hour s set at 500volt s (v) and 120 milliamperes (mA). Shorter running time led to lack of bands or poor separation of bands but longer duration allowed some bands to go overboard. The timing and constant voltage or current were maintained by a power supply (micro computer electrophoretic power supply, model E 455). The temperature in the tank was maintained at 3°C by a multi-temperature thermostatic circulator (LLK Bromma 2219). The use of bromophenol blue to monitor mobility was discontinued because it coagulated some samples. The running time of 4 hours was found to be adequate for
64 most enzymes because it produced good band separation and allowed gel assembly, running, staining and recording within the same day. Therefore, the need for storing them overnight was eliminated, as storage had negative effects on enzymes like PGI and SDH. At the end other run, the gels were carefully removed and stained appropriately. Enzymes such as GOT, ADH, MR and DIA took shorter time to stain. Longer exposure of these enzymes to the stain led to over-staining and heavy streaking. The electrophoretic phenotypes were scored and recorded in specially designed zymographs. Furthermore, some of the gels were photographed.
Preparation of extraction buffer Reagents Quantity fi-Mercaptoethanol 0.025 ml EDTA 0.010 g Potassium Chloride 0.019 g Magnesium chloride 0.050 g PVP 40 000 1.000g O.lMTris-HCl 25.000 ml
Preparation of acrylamide gels for electrophoresis analyses Stock solution: 1. Acrylamide stock solution: 63m l of distilled water was added to acrylamide/bis- acrylamide stock in a bottle, shaken well, marked and kept in a fridge. 2. Ammonium persulphate solution was prepared by dissolving 50 mg ammonium persulphate in 10 ml distilled water. Ammonium persulphate was prepared and used when still fresh. Poor results were obtained when stored ammonium persulphate was used.
Preparation of gel buffer The pH of the gel buffer was maintained at 8.4 by increasing (if low) with concen trated trizmabas e solutionan d reducing (if high)wit hdilut e a(50% )hydrochlori c acid solution. Reagents Quantity Boric acid 0.09 ml of 5.56g/l EDTA 0.0025 ml of 0.93g/l Trizma base 0.09 ml of 10.89g/l
Preparation of acrylamide gels Eight gels were prepared for each run. The acrylamide gels were prepared in the evening and used the following morning (day), allowing them time to form. The gels were prepared by mixing the solutions below:
65 Reagents Quantity Distilled water 48.00 ml Gel buffer 224.00 ml Acrylamide stock 88.00 ml Ammonium persulphate 21.60 ml Temed 0.72 ml
Preparation of tank buffers For each tank 5 litres of the buffer was prepared. In 5 litres of distilled water, the following chemicals were added: Reagents Quantity Boric acid 27.80 g EDTA 4.65 g Trizma base 54.45 g
A continuous buffer system of Tris-Borate-EDTA (Wendel & Weeden, 1989) was used. The pH was raised to 8.4 with trisma base (if low) and lowered with hydro chloric acid (if high). Polyacrylamide gels (16x18 cm) were prepared with a Hoeffer S.E. 600 apparatus for vertical electrophoresis. Using the accessory divider, four club gel sandwiches were put in each tank. Two tanks were used per run, with eight enzyme systems a total of twenty samples were screened per run (per day).
4.2.2 Staining procedures
The basic staining procedures for various enzymes for different taxa and tissues have been reviewed by Brewer & Singh (1970), Vallejos (1983) and Wendel & Weeden (1989). From these basic recipes, the optimum staining conditions was established for Vitexsample sb ymanipulatio no fpH , substrate concentrationan d buffer ionic strength. Tris-HCl staining buffer was used throughout the study. The staining reagents used besides the enzyme specific substrates were; tetrazolium thiazolyl blue (MTT), diazonium salts (i.e. fast blue BB salt, fast blue RR salt), co-factors such as nicotinamide dinucleotide (NAD), B-nicotinamidedinucleotid e phosphate (B-NADP), nicotinamide dinucleotide reduced (NADH),phenazin e methosulfate (PMS), acatalys t for most assays with tetrazolium stains. Other than NADH, the frequently used reagents were retained refrigerated as aqueous solutions of 10mg/m l and dispensed in 1 to 2 ml volumes. In order to mark the position of every sample during the staining procedure, a notch was cut at the top left hand corner of every gel next to the first specimen. A total of 15 enzymes were stained initially (Appendix 4.1), but only 8 showed consistently good resolution.
66 Results and discussions The enzyme systems that exhibited superior resolution of banding patterns were ADH, DIA, GOT, ME, MR, 6-PGD, PGIan d SDH (Table 4.1). They were thenuse d inth estudyin g genetic structureo fth e Vitexpopulations . Thebes tisozym e polymorph isms were observed in cotyledons of germinating seeds (seedlings). That could be attributed to high metabolic rates and enzymatic activities inth e developing seedlings. According to Smith (1981), cotyledons of seedlings are normally in an active state, mobilising resources for growth, thus they havehighe r levels of enzymes at that early stage of development. The use of germinating seeds also helped in avoiding dead or rotten seeds. The poor results from leaf samples could be attributed to the production of the photosynthetic substances associated with tannins, phenols and other inhibiting chemical by-products. Fresh leaves produce good bands with fewer enzymes (ADH, GOT, PGI and SDH) compared to seeds and germinating seeds. The activities were observed in leaves only when polyvinylpolypyrolidone (PVP 40 000) was included in the extraction buffer. That is likely to be a result of the ability of PVP 40 000 to render the inhibitors sucha sphenoli ccompound s inth esample s ineffective. Thebark s and roots exhibited heavily streaked bands or no activity with the enzymes screened. It is possible that better results can be achieved from the remaining enzymes and tissues of Vitexi f more screening is carried out, involving adjustment of extraction, staining and running conditions.
Table 4.1. Results from thetria lscreenin g ofdifferen t enzymes invariou sV. keniensis and V.fischeri tissues . The stainingprotoco l for each enzyme ispresente d in appendix 4.1.
Enzyme Plant material
seeds seedlings leaves roots bark
ACP * # * # * ** ADH + + + + + * # DIA + + + * # # ** a-EST * * ** # * GOT + + + + + + * * G-6-PD * * * * * GDH # + * * * LAP # # * * * MDH # * # # ** * ME + + + ** * * MR + + + ** * * 6-PGD + + + # * * PGI + + + + + * * PGM * + # * * SDH + + + + + * *
* No reaction; ** Streaking; + Activity, faint bands; + -I- Good, clear bands.
67 The analytical materials finally used in the study consisted of seeds from the 100 randomly selected trees in each of the four geographically isolated Vitexpopulation s inKenya , Uganda andTanzania . Sampleso fmatur e fruits were collected from allove r the crown, extracted by depulping, dried, cleaned and maintained separately for each of the selected trees. Theprocesse d seeds were maintained incol d storage at4° C until they were required for isozyme analysis. The genetic variation within andbetwee n the Vitex populations was studied by sampling one seed from each of the 100tree s selected per population. Therefore a total of 400 trees were sampled in the four Vitex populations for the study.
4.2.3 Isozyme analyses on samples of germinating seeds of V. keniensis and V. fischeri
Materials and methods 1. The processed seeds from the 100 sampled trees per population were rinsed in distilled water to remove dust and empty, rotten seeds by flotation. 2. Single viable seeds from each of the sampled trees were sown in vermiculite in a polystyrene sheet in a glasshouse, making a total of 100 seed samples per population. 3. After 10 days, the radicle started emerging and the germinating seeds were removed, their cotyledons excised and digested in extraction buffer for isozyme analyses.
The 8 enzyme systems which gave good enzyme polymorphisms and the banding patterns that could be scored from the seedlings are listed in Table 4.2, while their staining schedules are summarised and presented in appendix 4.1. The staining was carried out in stainless steel trays with lids in an incubator to keep off light. This is because some of the staining reagents are photosensitive.
Table 4.2. Enzyme systems chosen and used in the analysis of V. keniensis and V. fischeri populations.
Enzyme Abbreviation Commission number
1. Alcohol dehydrogenase ADH 1.1.1.1 2. Diaphorase DIA 1.6.99.- 3. Gluterate oxaloacetate transaminase GOT 2.6.1.1 4. Malic acid ME 1.1.1.37 5. Manadione reductase MR 1.6.99.- 6. 6-Phosphoglucose dehydrogenase 6-PGD 1.1.1.44 7. Phosphoglucose isomerase PGI 5.3.1.9 8. Shikimate dehydrogenase SDH 1.1.1.25
68 The staining intensity of theenzyme svarie d from onesampl et oanothe r irrespective of the population. The response of different tissues to assay conditions was also reflected in the resolution of the enzymes. During tissue preparation, ME could not withstand longer centrifugation but MR could. When the tissues were stored at -4°C, MR, 6-PGD and PGI activities were easily lost while ME, ADH and GOT remained active and could be assayed the following day. The apparent differences in the activities of the enzymes could be reflecting the temperature ranges at which they function. The enzymes that were active in both leaves and seeds exhibited no noticeabledifference s inbandin gpattern sbetwee ncotyledon so f germinating seedsan d fresh leaves.
Alcohol dehydrogenase (ADH)
The enzyme stained well with seedlings aswel l as fresh leaves but not with the root or bark samples. The bands from seed samples were relatively faint. The enzyme stainedfas t atincubatio ntemperatur e of37° Can dwa spron et oover-stainin g when left to stain for long. However, slight over-staining was desirable for clear photographs and band intensity since the gel background was transparent. When rinsed with cold water the bands lasted for a longer period of time. ADH was less sensitive to storage of the digested tissues overnight and omission of any of the buffer additives (such as PVP) did not cause any noticeable difference in the resolution.
Diaphorase (DIA)
DIA exhibited activity in samples of seeds, seedlings, leaves and bark. However, leaf and bark samples streaked heavily making it somewhat difficult to evaluate electrophoretic phenotypes in some cases. The best, clear banding patterns were observed in seedling material while those of the seeds were good but faint. In an attempt to make the bands robust a little more than 0.2 mg of 2,6-dichlorophenol- indolephenol was added but they soon spread out into each other, making them a little difficult to score. The gels were incubated at 37°C and over-staining was avoided by limiting the staining time. Theband swer e clearer whenth e excess stain wasrinse d off with cold water.
Gluterate Oxaloacetate (GOT)
GOT produced bands of good resolution with seeds, germinating seeds and leaves. It had activity with the roots and barks. Aspartic and a-ketoglutaric acid used in the assay of GOT have low solubility in the staining buffer, therefore the mixture had to be stirred and dissolved before the pH could be corrected to 7.25. The other staining reagents were added to the mixture when the gels were ready for staining. GOT showed stability and stained well even after overnight storage of extracts from the sampled plant tissues. The enzyme could be stained within a pH range of 7.25 to 8.0 but the bands were sharper at pH 7.25. Omission of any of the additives from the extraction buffer did not alter the resolution.
69 Malic Acid (ME)
ME displayed good activity and clear bands with seeds and germinating seeds but not with roots or bark. The leaves streaked heavily making it difficult to discern the bands. The intensity of staining of MEo n samples of seeds and seedlings was reduced when 2-B-mercaptoethanol or PVP was omitted from the standard extraction buffer. ME took longer to stain and the optimum pH for staining buffer was between pH 7.0 and 8.0. Only one zone showed clear activity at pH 7.0, increasing to two in some populations at pH 8.0. ME was stable and did not lose activity when stored overnight.
Manadione reductase (MR)
The enzyme produced good clear bands with seeds and seedlings but streaked heavily with leaf samples. It displayed low activity after storage of tissue extracts overnight. No activity was observed in roots and bark. The effects of omitting some additives from the extraction buffer was not conspicuous. MR stained fast at 37°C and is also prone to over-staining. It streaked heavily when left to over-stain.
6-phosphogluconate dehydrogenase (6-PGD)
It produced no activity with leaves, roots or bark but seed samples exhibited faint bands. The best resolution was observed on samples of germinating seeds (seedlings). In the preparation of its staining reagent, 6-phosphogluconic acid was first dissolved in staining buffer and the pH corrected to 8.0. The enzyme exhibited stability but required longer staining time to allow slight over-staining for clear resolution. However, it mostly displayed one zone of activity but two in some rare cases.
Phosphoglucoisomerase (PGI)
PGIdisplaye d goodactivit yespeciall ywit h seedlings, seedsan d leaves. Theactivit y on seeds was a little faint. It stained slowly, therefore it required longer in staining reagent. Good resultswer eobtaine d whenth e stainingproces s wasallowe d tocontinu e over night. There was no activity in roots and bark extracts.
Shikimate dehydrogenase (SDH)
Theactivit y andresolutio n datawer eobtaine d from extracts ofseedlings , leavesan d seeds. The bands from seed samples were faint but they could be scored. SDH was dissolved in staining buffer and pH corrected to 8.0 before mixing with the other reagents. The enzymewa sstabl ebu t stained slowly thusneede d longer stainingperio d in an incubator at 37°C.
Conclusion The observations made seem to be consistent with the general conclusion that the range of variables for good electrophoretic resolution are enormous, and the optimum
70 conditions can only be searched empirically. The variation that could be analyzed is represented by the isozyme patterns that account for the number of bands, their distribution and staining intensity, but the probability of experimental artifacts is also high until sufficient samples are assayed and analyzed genetically. The following chapter presents the genetic interpretation of the isozyme patterns observed inth e gels run under the conditions which were optimised in the screening runs.
Following the above protocol the eight enzymes (ADH, DIA, 6-PGD, ME, MR, GOT, PGI and SDH) are suitable for genetic analysis on Vitex. The best tissues are from germinating seeds and to some extent fresh leaves (Table 4.1). The good results from germinating seeds could be attributed to high metabolic activity, resource mobilisation andthu shig h enzyme activities. Storage oftissu esample s isno t advisable as most of the enzyme systems lose activity when stored. However, failure of most of enzyme systems to produce bands with leaves of bark could be attributed to low enzyme activity or thepresenc e of tannins, phenols and other inhibitors in those parts of the plant. The obstacle could be overcome by further research in inhibitor neutralising substances.
4.3 Electrophoretic analysis on polyploidy and allozyme polymorphism in V. keniensis and V. fischeri
The ploidy level of a given species can be obtained from chromosome counts, chromosome pairing pattern andmorphologica l variation, butmor e recently, isozymes and other molecular techniques have become useful in determining ploidy levels (Harris & Ingram, 1992). This chapter presents an isozymic assessment of the level and nature of the polyploidy of the different the V. keniensis and V. fischeri populations in East Africa. The information is also valuable in interpretation of complex electrophoretic data that arise from increased ploidy level (Wolf et al., 1990; Barrington, 1990; Haufler et al., 1990).
The techniques and the results of chromosome counts are presented in chapter 2. The survey showed that all the Vitex populations studied had one cytotype of 96 somatic chromosomes. Despite the variability inth e ecology of the zones occupied by the different Vitexpopulations , the species seem to have retained the same cytotype. However, the chromosomes were too tiny for the details of karyotypes to be studied. The analysis of allozymic pattern was used to reveal the segregation and inheritance pattern, and the nature of the polyploidy (auto-, alio- or segmental polyploid). The interpretation of allozymedat ai sdirectl y affected byth especies ' ploidy level, andthu s the basic understanding of the species' cytotype is necessary (Barrington, 1990; Haufler et al. 1990).
The application of enzyme phenotypes to ascertain ploidy level has been demon strated for many plant species (Crawford & Smith, 1984; Haufler et al., 1990). In all these casespolyploid s were shown todispla y isozyme multiplicity relative todiploids .
71 Through the allozyme data, the nature of the ploidy can be determined since multisomic inheritance is mostly associated with autopolyploids, while fixed heterozygosity is a character of allopolyploids (Waples, 1988; Krebs & Hancock, 1989; Cai &Chinnappa , 1989). Theinheritanc epattern s of allozymesca nals ob euse d to identify thestatu s of aspecies ' cytotype (Odrzykoski &Gottlieb , 1984). Thiswoul d allow the distinction between auto-, alio- and segmental polyploids. Electrophoretic characteristics associated with multisomic inheritance include the number of loci displayed, asymetrical banding patterns, fixed heterozygosity, and multiple alleles within the same locus (Barret & Shore, 1989; Wendel & Weeden, 1989; Kephart, 1990). Allopolyploids tend to exhibit fixed heterozygosity at many loci due to non- segregation of non-homologous chromosomes (Werth et al., 1985).
This chapter examines the patterns of inheritance in electrophoretic loci of V. keniensisan d V. fischeri. The results were eventually applied in the scoring, analysis and interpretation of the genetic data from the 8 enzyme systems on the four Vitex populations, covering the geographical range of the species. Inferences are drawn about the possibility of higher level of ploidy, its nature and genetic polymorphism from the pattern of allozyme inheritance.
Materials and methods 1. The isozyme genotypes of five of the ten parent plants used in artificial pollination experiments (see chapter 3) were analyzed. 2. Seeds from emasculated, selfed and bagged flowers (treatments 3, 4 and 5i nth e pollination experiments) enough to raise 40 progenies per selected tree in each population were sown in vermiculite in a germination cabinet at 25°C . 3. Young actively growing leaves were collected the seedlings at the age of 3 months. 4. The protocol of isozyme analysis is explained above. When more than one loci were observed for an enzyme, the most anodally migrating locus was designated as 1. The alleles were given letters with the most anodally migrating one designated as a, the second fastest b, and so on. 5. Four enzyme systems (ADH, GOT, PGI and 6-PGD) were tested using Vitex seedling leaf extracts. These were the only enzyme systems with relatively good resolution on Vitexlea f extracts. 6. Atota l of 800progenie s were analyzed covering the four Vitex populations. The banding patterns of the progenies were scored and compared with the parental phenotypes. 7. The segregation ratios ofprogenie s were studiedfro m electrophoretic phenotypes and were compared with the expected ratios derived from the genotypes of the parent plants.
Results and discussions The enzyme electrophoresis resulted in a clear staining for the four enzymes encoded by 6 loci; ADHl5 ADH2, GOT2, PGI, PGI2. and 6-PGD2. Most of the plants exhibited these polymorphic loci. The bands migrated anodally for all the examined
72 enzymes. Several progenies exhibited heterozygous banding patterns at the different enzymes andwer ehighl ypolymorphic . Somedat ao nth esegregatio n amongprogenie s of the Vitexar e presented in table 4.3. Most loci displayed three to four bands per locus for mostenzyme s andther ewa s noevidenc efo r fixed heterozygosity. They also exhibited unbalanced staining intensities in a number of enzyme loci, a possible indication for multiple doses of individual alleles. Genotypic interpretations of electrophoretic band patterns were inferred by assuming that banding intensity reflect allelic dosage. The segregation atth e six enzyme loci was inclos e agreement with the genetic model of tetrasomic inheritance, codominant expression of alleles coding for enzymes and random chromosome segregation (Table 4.1).
These observations are evidence for tetrasomic inheritance in all the populations of V.keniensis an d V. fischeri, enabling the species to maintain up to 3 to 4 alleles at a locus. However, complex banding patterns for ADH2 consisting of up to five bands were also observed occasionally in some plants; these could be heterodimeric bands of intermediate mobility at times formed between the three allozymes (Soltis & Rieseberg, 1986). There was no evidence of fixed heterozygosity which is a predictable and consistently observed consequence of allopolyploidy (Gotlieb, 1981; Werth et al., 1985). In all the polymorphic loci, both homozygous, balanced and unbalanced heterozygous banding patterns were displayed in progenies. A number of electrophoretic investigations onallopolyploid s haveconsistentl y revealed thepresenc e of fixed heterozygosity (Soltis & Rieseberg, 1986) arising from lack of segregation. Therefore, the observed homozygosity, normal heterozygosity and complex banding (independent segregation) patterns in the progeny arrays is an evidence for autopoly- ploidy in the species. While, the maximum of four alleles per locus with occasional exceptions discussed above implies tetraploidy. The species may therefore be considered an autotetraploid. The results from Vitex is similar to those of obtained by Quiros (1982), Crawford & Smith (1984) from a number of taxa which showed that autotetraploid individuals carry up to four different alleles per locus.
Conclusion The data from isozyme analysis on electrophoretic phenotypes and segregation in progeny arrays of the Vitex populations has provided additional insight into the nature of polyploidy in the plant species. The populations of Vitex exhibited autotetraploidy; as deduced from the 96 somatic chromosomes, enzyme multiplicity, the maintenance of up to three or four alleles at a locus and tetrasomic segregation pattern in the progenies (Table 4.3 &Figur e 4.1). The independent (random) segregation of the four homologous chromosomes during the first meiotic division tends to lower the frequency of homozygous gametes (Krebs & Hancock, 1989) and that could explain the observed heterozygosity among the progeny of Vitex.
In general the tetrasomic inheritance results in a high proportion of heterozygous progeny as reflected inhig h levels of heterozygosity in autotetraploid populations (Li, 1955). It has been suggested that enzyme multiplicity affords a polyploid greater biochemical versatility (Barber, 1970; Manwell & Baker, 1970; Levin, 1983).
73 Table 4.3. A n example of segregation
ADH,: aabc x aabc ADH2: bccc x bccc
Progeny Expected Observed Progeny Expected Observed
aaab 4.4 5 bbcc 10 12 aaaa 1.1 2 cccc 10 9 aaac 4.4 2 bccc 20 19 aabc 11.1 13 X2 0.55ns aabb 4.4 5 abbe 4.4 4
aacc 4.4 4 6-PGD2: bbcc x bbcc abec 4.4 3 Progeny Expected Observed bbcc 1.1 2 2 X 2.92ns bbbb 1.1 2 bbbc 8.9 11 bbcc 20.0 19 GOT2: aabc x aabc bccc 8.9 8 cccc 1.1 1 Progeny Expected Observed X2 1.03ns aaac 4.4 3 aaab 4.4 6
aaaa 1.1 1 PGI2: acef x acef aabc 11.1 12 Progeny Expected Observed aabb 4.4 3 abbe 4.4 5 ceef 2.2 3 aacc 4.4 5 ceff 1.1 1 abec 4.4 4 aacc 1.1 0 bbcc 1.1 1 aace 2.2 2 2 X 1.7ns aacf 2.2 3 acce 2.2 3 accf 2.2 2 PGI,: bbce x bbce ceff 2.2 1 Progeny Expected Observed acef 6.7 8 aaee 1.1 1 bbbc 4.4 6 aaef 2.2 2 bbce 11.1 11 acee 2.2 1 bbbe 4.4 4 eeff 1.1 0 bbbb 1.1 2 aeef 2.2 4 bbcc 4.4 5 aaaf 1.1 2 bece 4.4 4 aeff 2.2 3 bbee 4.4 3 aeff 2.2 2 bcee 4.4 4 ccee 1.1 0 ccee 1.1 1 ccef 2.2 2 X2 3.2ns X2 2.82ns
Note: The example given is at each of the six loci in an array of progenies from one of the selfed trees. The statistically significant differences between the expected and theobserve d number of genotypes were analyzed by chi-square test, X2 (p < 0.05 and p < 0.10). Thedifferenc e between theexpecte d andth eobserve d number ofprogenie s was insignificant in all the crosses (ns) both at p<0.05 and p<0.10.
74 fc'-:-:-HlW
V -*•}?'..$¥ ^
_ _ im • i ** *» •*. *i ^ ^» ". V "*•
Figure 4.1. Photographs of polyacrylamide gels (PAGE) showing electrophoretic patterns and tetrasomicsegregatio n ina narra y of 20 Vitex progenies from selling. The numbers at the right of the photographs designate loci and alleles, respectively. Numbers at the bottom indicate individual samples. A. ADH (ADH^ & ADH2); homozygotes (one banded), normal heterozygotes, unbalanced heterozygotes and complex heterozygotes. Note the 5 band heterozygotes in ADH2. B. GOT2; homozygous(on eband) ,norma lheterozygous ,unbalance dheterozygous .Three-bande d patterns were also depicted. C. PGI2; homozygous (one-band), normal heterozygous, unbalancedheterozygous , three-bandedan dcomple xpatterns .D .6-PGD 2;homozygou s (onebanded) , normal heterozygous, unbalanced heterozygous andthre eban dpatterns .
According to Soltis & Rieseberg (1986) the multiple enzyme forms may minimise variation in substrate affinity, thus expanding the range over which physiological coordination of metabolic processes is possible. The observed heterozygosity and enzyme multiplicity may therefore be an adaptation by the species for greater genetic and biochemical versatility, a possible explanation for the wide ecological range of natural occurrence (i.e Savanna and montane rain forest) by the Vitex populations.
4.4 Electrophoretic phenotypes in V. keniensisan d V. fischeri populations
Inorde r todetermin e theelectrophoreti c (genetic) relationshipwithi nan damon gth e four different Vitex populations, all the 8 enzymes assayed were scrutinised for variation in their banding patterns (Figures 4.2 & 4.3).
75 , n •• «KM •> 1 %'•P2Z " " «
T'l J • •• '
I.'
' •• •} -. ?•
i * * *•««.* »;..^ (i* (i,t »j» " w %i * (J n ao
Figure4.2 . Electrophoretic phenotypes ofV. keniensis andV. fischeri (isozym e loci): A. GOT2 & GOT3; B. ADH„ ADH2 & ADH3; C. DIA2; D. 6-PGD2.
Twelve loci expressed bandingpattern s that could reflect thegeneti c structure ofth e study species' populations. Theenzyme swer edesignate d according toth e international nomenclature followed by arabic numbers, withth emos t anodal designated as the first locus. All the assayed enzymes migrated anodally, and the number andpattern s of the bands which contributed to a locus were inferred from the sub-unit structure of the enzyme as described by Wendel &Weede n (1989), Kephart (1990), Crawford (1990) and Murphy et al. (1990). Alleles at a locus were specified by lower case letters following Cai and Chinnapa (1989) and Shore (1991). Where two loci migrated to the samepositio n on agel , thepossibl e homoelogous alleles were assigned similar letters.
To confirm that the migration of allozymes at a locus was similar, samples from various populations were run adjacent to each other on the same gel and, in several combinations, for allth eenzymes . Progenies (selfed) from parentso fknow n genotypes were analyzed to reveal the species' allelic segregation (Table 4.3). The phenotypes were therefore scored into genotypes, assuming that the variation in banding patterns reflected allelicpolymorphism , describeda sth eoccurrenc e together inth esam ehabita t of two or more discontinuous forms of a species (trait or allele) in such proportions that the rarest of them cannot be maintained by recurrent mutation (Ford, 1976). The ratios of the various genotypes at every locus, for all polymorphic loci, to the total sample size were then expressed as genotypic frequencies. The populations were then entered into comparative analysis.
76 6
•» * ' * 3 : -" * i a. a A.s J»«I '«i«nfijjj
^.^
******4-- ,*2.sRL .
UjtS^ * * 1 lo 11n (l Kj i
Figure 4.3. Electrophoretic phenotypes of V.keniensis an d V.fischeri: A . SDH,; B. ME,; C. PGI, & PGI2; D. MR,.
The electrophoretic phenotypes were then scored into genotypes. The ratios of the various genotypes atever y locus, for allpolymorphi c loci, toth etota l sample sizewa s expressed as genotypic frequencies. The distinction in the allelic frequencies was compared with ecological conditions of each population. The 12 loci analyzed were GOT2, GOT3, Adh„ ADH2, ADH3, DIA2, MR,, SDH,, 6-PGD2, PGI2, PGI3an d ME, (Table 4.2) for enzyme nomenclature and commission number. The observed electro phoretic patterns of the various enzymes and their interpretation are discussed below.
Gluterate Oxaloacetate (GOT)
The Vitex populations showed three zones of activity in GOT (Figure 4.2 A). However, the last two zones (loci) were consistent and clear for analysis. They were designated GOT2 and GOT3. The first locus (GOT,) showed no variation between the trees and even populations. GOT2 exhibited complex pattern, with some genotypes displaying amaximu m of 4allele s withvaryin g degreeo f staining intensity in different samples. There were two alleles which could appear either as a pair or independently in the locus. That was noted in anumbe r of alleles in the locus. The complex banding pattern observed in GOT2 could have resulted from overlapping of two loci co- migrating on a gel. The presence of some homozygous genotypes assisted in maintaining the interpretation on the possibility that locus GOT2 was a reflection of multiple alleles within the locus. GOT2 and GOT3 were predominantly expressed by
77 three alleles per locus with asymmetrical staining. The some of the samples exhibited four alleles per locus with asymmetrical staining intensities.
A secondary band activity cathodal to locus two was noticed in some samples but itsgeneti c significance was notestablishe d because itwa svariabl e inintensit y and was not consistent with any sub-unit structure. The observation was consistent with the activitiestha tha dbee nreporte d onStephanomeria exigua (Gottlieb, 1973),whic hwer e referred to as 'ghost bands'. These bands could be a characteristics of some enzyme systems Kephart (1990) or they could be representing some silenced alleles. Their activities varied among individuals within the populations, possibly implying some inheritance pattern.
Alcohol dehydrogenase (ADH)
The enzyme exhibited three zones of activity in the samples of Vitex. The anodal locus was designated as ADHl5 the cathodal zone, ADH3 and the middle ADH2. The three zones of activities exhibited some variation within and between the populations. The pattern and number of bands varied considerably between the activity zones. Locus ADH2 showed the highest number of bands. In some samples, as many as 8 bands were observed atADH 2 in some samples. ADH!an d ADH3displaye d relatively fewer bands and less variation than the middle zone (ADH2). This could be attributed to ADH being a dimeric enzyme. It is therefore expected to form intragenic heterodimers inheterozygou s situation(Hart , 1969, 1970;Marsha l etal. , 1974;Brow n etal. , 1974;Roos e &Gottlieb , 1980). According toGottlie b (1982), theabilit y oftw o alleles to form heterodimers is a sign of structural similarity. Thus the likely reason for alleles a, b, c, d and e at ADH2 to selectively form heterodimer bands could be based on their structural relationship (Figure 4.2 B).
Diaphorase (DIA)
DIA had 2mai n zones of activity (loci). Theanoda l DIA2wa s clearer and exhibited polymorphism which was included in the study of the populations while DIA! was monomorphic in most samples. DIAj exhibited up to 4 bands but the majority of samples displayed 3 alleles (Figure 4.2 C). The enzyme is known to display considerable variation from one species toanothe r in terms of the enzymes expressed, and quaternary structure. Inreview sb yWende l &Weede n (1989) and Kephart(1990) , the enzyme is presented as a monomer, a dimer or a tetramer. DIA is one of the reductase enzymes which are not substrate-specific (Wendel & Weeden, 1989), and thus may stain other reductase enzymes. That was not a common occurrence but ina few isolated cases some bands resemble MR. But parallel staining of DIA and MR on the same day assisted in eliminating the stray bands.
6-Phosphoglucose dehydrogenase (6-PGD)
6-PGD is one of the enzymes of the carbohydrate metabolism which is frequently
78 assayed inplan t species. In V.keniensis and V.fischeri, 6-PG D displayed 2 loci. The anodal was designated 6-PGD! and the cathodal one 6-PGD2. Of the two loci, 6-PGD2 was the most consistent and polymorphic. 6-PGD! was rare and was mostly monoallelic whenever it occurred (Figure 4.2 D). For 6-PGD2 up to 4 alleles were observed per locus but 3 alleles were more common. Some alleles stained darker, especially when the b allele was present. That could be expected since the enzyme is a dimer (Wendel & Weeden, 1989; Kephart, 1990; Murphy et al., 1990). In phenotypes with three bands, deep staining was also observed; this could be a reflection unbalanced heterozygosity. The staining intensity of four bands varied. That could be interpreted as dosage effect and further differentiated samples. 6-PGDi is usually (not always) located in cytosol, while 6-PGD2 is a plastid allozyme (Oballa, 1993). Theenzym ei shighl y conservativean di susuall yfoun d astw oisozymes ,excep t in case of duplication (Gottlieb, 1981; 1982). The bands displayed asymmetrical staining and unbalanced heterozygosity (Figure 4.2 D).
Shikimate Dehydrogenase (SHD)
SDH, displayed one zone of activity with up to four bands, representing 4 alleles, designated from a to d. SDH is a monomer (Wendel & Weeden, 1989; Kephart, 1990), displaying single bands for homozygotes and, two to three bands for heterozygotes. Thesample sexhibite dbot hhomozygosit y andheterozygosit y (balanced and unbalanced). Some genotypes exhibited four alleles which migrated closely together while in others were the alleles were slightly separated but the pattern maintained. Genotypes with three bands were common inth e four populations (Figure 4.3 A).
Malic Enzyme (ME)
ME is also known as MDPH (Murphy et al., 1990). It exhibited 2 enzyme activity zones (Figure 4.3 B). Theanoda lwa sdesignate d MEi andth ecathoda l ME2. ME2wa s mostly monomorphic and inconsitent in the samples. The phenotypes in ME, had variable number of bands ranging from one to four. The one-banded phenotypes were noted as homozygotes, and the remainder with two, three or four were considered heterozygotes. The three- to four-banded phenotypes were classified as balanced or unbalanced heterozygotes depending on the gene dosage effect and the patterns. Murphy et al. (1990) and Kephart (1990) described ME as a tetramer. However, the genotypes were a little difficult to score due to unclear sub-unit structure. The less clear banding patterns and sub-unit structure of the enzyme could be a possible expression of multiple bands, and variable dosage ratio which is only possible in polyploids (Oballa, 1993).
Phosphoglucoisomerase (PGI)
Two regions of activity, PGI, and PGI2 were observed in the enzyme. Both exhibitedu pt osi xallele spe r locus(Figur e4. 3C) . Thecomple xbandin gpatter ncoul d
79 be attributed to co-migration of the alleles. (Figure 4.3 C). The enzyme was highly polymorphic and displayed variation between the species' populations.
Generally, the polymorphism varied from one locus to another but the locus that displayed high number of alleles also showed high number of genotypes. For instance ADH2 showed the highest number of alleles compared to other loci, meanwhile SDH; had the lowest number of alleles and polymorphic genotypes.
Manadione Reductase (MR)
MR exhibited one zone of activity zone, designated as MR^ The activity zone had variable bands confirming theexistenc eo fhomozygote s andheterozygote s (Figure 4.3 D). The enzyme hasbee n reported to express tetrameric banding patterns by Bousquet et al. (1987) and Huenneke (1985). Heterozygotes expressed up to four bands and homozygotes one. Somesample s displayed patterns withthre e intensely stainedbands . This could have resulted from interaction factors when heterodimers co-migrate or individual variation in activity of the enzymes. However, it could also represent multiple alleles expressed by homologous chromosomes. The difficulty experienced was the sensitivity of the enzyme to assay conditions leading to some inconsistent variability.
4.5 Genetic diversity of V. keniensisan d V. fischeri populations
The pattern of genetic diversity can be inferred from isozyme markers (Ledig, 1986). It provides an insight into historical biogeography of populations, revealing the past geographic structure and distribution (Ranker et al., 1994). Several investigations of the level of genetic diversity among congeneric species involving diploids and their polyploid counterparts have indicated that polyploids do exhibit higher heterozygosity than diploids (Schnabel et al., 1991). About 40% of plant species are known to be polyploids, and most of these are angiosperms (Stebbins, 1980). Evidence from cytological and isozymepattern s shows thatV. keniensis and V.fischeri ar e polyploids (bothhavin g 2n=96), thus raising theexpectation s of observing ahig h levelo f genetic variation.
Isozyme genemarker s havebee nfoun d tob establ ean dreliabl e indicatorso f genetic variation and population structure (Nei, 1972; 1973; 1978; Gottlieb, 1981; Adams, 1983; Hillis & Moritz, 1990) because of their direct link to genes. This Chapter addresses the genetic variability within and between the different Vitexpopulations . Twelve polymorphic isozyme loci of 400 samples from four populations were used to determine the variation of genetic parameters. The possible taxonomic distinction and evolutionary trend of the populations of the species are also illustrated using dendrograms. The application of the observed genetic variation in conservation strategies and breeding programmes are also discussed.
80 4.5.1 Statistical analysis on the electrophoretic data
The 8 enzymes examined resulted in the identification of 12 putative loci which were used the genetic analyses. The banding and segregation pattern confirmed autotetraploidy in the species. In addition to homozygotes, balanced and unbalanced heterozygotes were also evident. Such observations are normally a consequence of different allelic dosages (Soltis & Rieseberg, 1986; Ehrendorfer et al., 1996) which couldhav earise nfro m tetraploidy.Th ebandin gpattern s(phenotypes )wer escore dint o genotypes and further transformed into relative genotypic frequencies by counting individuals of similar genotypic categories and calculating the ratio to the total sample size. Appendix 4.2 the allelic frequencies at the 12 loci of the Vitexpopulations .
The parameters expressing genetic variation were calculated both for the species as a whole and individual populations (Table 4.4 & 4.5). The proportions of the polymorphic loci (P) and the mean number of alleles per locus (A) were calculated using the computer program BIOSYS-1 (Swofford & Selander, 1981). The allelic frequency, effective number of alleles and expected heterozygosities were calculated manually. Other genetic parameters were estimated following the formulae of Nei (1973, 1975) and Hedrick (1985). These included total gene diversity (HT), expected geneheterozygosit y (He),intrapopulatio ndiversit y (Hs),inter-populatio ngen ediversit y (Dst), the coefficient of gene differentiation (GST), the coefficient of absolute gene differentiation (DM) and the coefficient of intra-population gene diversity (RST)- Nei's (1978) unbiased genetic identity (I) and genetic distance (D) were calculated using BIOSYS-1. The rooted Wagner Tree dendrogram was also plotted byBIOSYS-1 .
4.5.2 The proportion of polymorphic loci (p)
The proportion of polymorphic loci (P) is one of the parameters used to determine genetic variation in natural populations using isozyme loci polymorphism. It is a statistic expressing the number of loci with two or more alleles. The parameter for each Vitex population was estimated following Hedrick (1985), where xi s the number of loci polymorphic in the sample and m is the number of loci assayed, including monomorphic loci:
P = - (1) m
The average polymorphism for the species was then calculated as follows, where s is the number of populations of the species examined:
p = 1= 1
81 4.5.3 Estimates of genetic diversity of populations of V. keniensis and V. fischeri
The isozyme phenotypes were scored into genotypes and further transformed into genotypic frequencies by counting individuals with similar genotypes and calculating their ratio to the total sample. The observed mean heterozygosity (H0P) in each population was calculated as follows:
„ EG £"_ (3) H OP m where N is the sample size of apopulation , Gi sth enumbe r of observed heterozygous genotypes and m is the number of loci scored, including the monomorphic ones. The overall observed mean heterozygosity for the species (Hos) was then estimated by averaging over the populations:
£ H°Pi (4) Hos = J—~ where s is the number of populations, other terms are defined in formula 3.
Thegen ediversit y ofth especie swa scalculate d assuming autotetraploidy (presented in section 4.4) and that the populations were randomly mating i.e. Hardy-Weinberg equilibrium. In a homozygote of an autotetraploid, the four alleles are identical. Therefore for those isozyme loci with the four co-migrating alleles were considered homozygotes. The estimate of the expected heterozygosity was calculated using the formula of Hedrick (1985) for autotetraploids, as:
He - 1- tf! (5) ;=i where n is the number of alleles and pi4 is the frequencies of the homozygotes.
The effective number of alleles (Aep) in apopulation , which is the reciprocal of the frequency of the homozygotes, was calculated following Ayala and Kiger (1984), as (all terms are as defined above): I K 4 (6)
Ef1= 1 t
82 The average expected heterozygosity (Hep) of the populations was estimated as the average of the per locus heterozygosity estimates:
Eff«i (7) M H -_ 1 = 1 ep = m
The genetic diversity within the species at each locus (HSP) was calculated as (all terms are as defined above):
s I>i (8)
The overall average genetic diversity within the species' populations (Hss) is then;
m HHsPi (9) #„ = — m
The species' total genetic diversity, HT at each locus is calculated as
m n E^E'4' do) H _ i=i i=i ii j — m where r is the weighted allele frequencies over spopulations .
Inter-population genetic diversity (DST) is the absolute measurement of genetic differentiation among populations (Nei, 1973; 1987), and was calculated as follows:
DST = HST - Hs (11)
The parameter above is a modification of Wright's F-statistic (Wright, 1965), adapted to allow analyses of loci with multiple alleles, because the original formula, [1 -Fit=( 1-Fis)( 1-Fst) ] could not handle a situation where more than two alleles are expressed ata locus (Nei, 1973). Fit, Fis and Fstar edeviation s of genotypes from the Hardy-Weinberg equilibrium, which is more complex when applied to polyploids (Oballa, 1993). The complexities such as overlapping alleles leading to either under- or over-estimations of genotypic frequencies in multi-allelic polyploids with homo logous genes have been discussed by Asins & Carbonell (1987) and Waples (1988).
83 Nei (1973; 1975)define d the absolutemeasur e of geneticdifferentiatio n (DM)a sth e averageminimu m geneticdistanc ebetwee npopulations , and it isdependen t ongeneti c diversities withinpopulations . Itexclude s comparisons of populations with themselves and is appropriate for comparing genetic differentiation in different populations. It is estimated as (all terms are as defined above): sD D = ^ST (12) M s-1
The coefficient of genetic differentiation (GST) is an index expressing inter- population variation relative to total diversity, and was estimated as:
13 GST = ^ < >
The above estimate of GST is useful when comparing populations with similar breeding systems (Nei, 1973). It varies from 0 to 1an d is equivalent to Wright's Fst (gene differentiation among populations), as demonstrated by Nei (1973).
Coefficient of intra-population gene diversity (RST) is the index of genetic diversity due to absolute gene differentiation amongpopulation s (Nei, 1973). The coefficient of intra-population gene diversity was calculated from Dm (Nei, 1973), as (all terms are as defined above):
RST =^ (14)
Totalgeneti cdiversit y (HT), intra-population geneticdiversit y (Hs), inter-population genetic diversity (DST), coefficient of geneticdifferentiatio n (GST), absolute coefficient of genetic differentiation (DJ and coefficient of inter-population genetic diversity (RST). The allele frequencies of each of the populations at the 12 polymorphic loci (Appendix 4.2) are also estimated.
Results and discussions The proportion of polymorphic loci is high (P= 100%) but could be considered comparable to other tropical tree species reported; 95% for Acaciatortilis (Oln'gotie, 1992), 90% for Faidherbia albida(Jol y et al., 1992), 98% forAcacia karroo (Oballa , 1993) and 66.7% for Abronia macrocarpa(Williamso n & Werth, 1999). The high polymorphic value could be attributed to either the species' biological characteristics such as long life cycle, high ploidy and out-crossing level. However, it is worth mentioning that overestimation of polymorphism can also arise from deliberate exclusion of non-consistent, poorly resolved monomorphic loci from the analysis.
84 Table 4.4. Distribution of genetic diversity estimates for the four populations of V. keniensis and V. fischeri. Sample size (N), mean number of alleles per locus (A), effective number of alleles (Aep), percentage polymorphic loci (P), observed heterozygosity (H0) and expected heterozygosity (Hep).
Population N A Aep P Ho Hep
Mt. Kenya 100 5.1 6.0 100 0.86 0.971 Nyambene 100 5.1 4.4 100 0.81 0.970 Mt. Elgon 100 5.1 6.4 100 0.87 0.971 L. Victoria 100 5.1 4.2 100 0.80 0.970
Species mean 5.1 5.3 100 0.84 0.970
Table4.5 . Genetic differentiationa t 10loc i acrossth efou r V. keniensis andV .fischeri 5opulations.
Locus HT Hs DST Gst Dm Rst ADH, 0.99 0.967 0.023 0.023 0.007 0.007
ADH2 1.00 0.965 0.035 0.035 0.012 0.012
ADH3 1.00 0.967 0.033 0.033 0.011 0.011 DIA2 0.98 0.965 0.035 0.035 0.012 0.012
GOT2 1.00 0.967 0.037 0.037 0.012 0.012 GOT3 0.98 0.967 0.037 0.037 0.012 0.012 MR, 0.99 0.963 0.037 0.037 0.012 0.012 ME, 1.00 0.962 0.038 0.038 0.013 0.014
SDH2 0.99 0.959 0.041 0.041 0.014 0.015 6-PGD, 0.98 0.965 0.035 0.035 0.012 0.012 PGI, 1.00 0.972 0.028 0.028 0.009 0.009
PGI2 0.99 0.969 0.031 0.031 0.010 0.010
Species mean 0.99 0.965 0.035 0.035 0.012 0.012
The enzymes were polymorphic for three or more alleles per locus. The effective number of alleles per locus (Aep) in the populations ranged from 4.2 to 6.0. While the mean number of alleles per locus (A) over thefou r populations was 5.1, this is similar in all the populations studied. Examples of reports on high average number of alleles per locus are Pinus taeda (3.89 to 4.78, Conkle, 1981) and Madura pomifera (4.0, Schnabel et al., 1991). The high number of alleles per locus and polymorphisms in Vitex could alsob e attributed toth epolyploidy . The constant number of alleles despite the variation in geographical and ecological conditions canals ob e a result of common origin, biological capacity of the populations to inhabit the different ecological zones without undergoing adaptive changes or the isozymes may be neutral to the forces of selection imposed by the environment, thus remaining unchanged over generations.
Generally, some angiosperms have higher levels of genetic diversity than those reported in conifers (Hamrick et al. 1981). The following are reports on the levels of the genetic diversity on some tropical angiosperms; Acacia albida45 % (Joly et al.,
85 1992) and Acacia tortilis (Oln'gotie, 1992), 73% for Madura pomifera (Schnabel et al., 1991), 83% for Acacia karroo (Oballa, 1993) and 99% for V. fischeri (= V. keniensis) in this study.The high level of genetic diversity in V. keniensis and V. fischeri populations could be attributed to the polyploidy and the widespread pollen dispersal by bees, and seed dispersal by monkeys and hornbill birds within the populations. Most studies comparing genetic diversity indiploid s and polyploids have also concluded that high variation is associated with high ploidy level (Shore, 1991). The attributes of polyploidy dominate the development and maintenance of genetic variation in a polyploid species (Arft & Ranker, 1998).
Theobserve d heterozygosity (H0) ranged from 80% inLak eVictori a to 87% in Mt. Elgon. The observed mean heterozygosity was 84%whil e theexpecte d heterozygosity (He=97%) was higher than the observed heterozygosity. Estimates of other genetic diversity parameters are alsopresente d in tables 4.4 &4.5 . The total genetic diversity of the populations based on the 12 loci (HT) was 99% while the intra-population heterozygosity varied from (Hs) varied from 95.9% (SDH2) to 97.2% (PGIj) with the mean of 96.5%. This indicates that thebul k of the species' genetic diversity isretaine d within the populations and little is distributed between them.
Themea n diversity among thepopulation s (DST)wa s estimated as 0.035. This value issmall , anindicatio n thatth e Vitexpopulation s areno tsignificantl y different from one another. The value of DST varied from 0.023 in ADH, to 0.041 in SDH2. The proportion of between population heterozygosity to the total heterozygosity (GST) was 3.5%. The GSTvalu e is used to compare the ratio of the population component to the total diversity. This implies that 3.5% of the total genetic diversity was attributed to genetic differences among the populations and the remaining (96.5%) was maintained within the populations of the species. The low GST value indicated that the Vitex populations are very similar, this is surprising given the disjunct ecological and geographical distribution of itspopulations . However, this mayb e afurthe r indication of taxonomic similarity between them as earlier presented in chapter 2.
High level of genetic diversity within the populations is mostly caused and maintained by an efficient gene flow and high fecundity (Hamrick et al., 1981; Loveless & Hamrick, 1984; Hamrick & Godt, 1989). Given the sedentary nature of trees, geneflow does occur through pollen and seed dispersal. This maintains and even increases the diversity of the genepool in a population. The high genetic diversity of the Vitex populations could beattribute d toefficien t gene flow through bee pollination and seed dispersal by monkeys and hornbill birds. This also suggests that although the tree species exhibits amixe d mating system, outcrossing seem tob e predominant thus the maintenance of the high genetic diversity within its populations. According to Barrett &Shor e (1989) observed that selfing plants tend tohav emor e genetic variation between than within their populations. Hamrick & Godt (1989) also found that trees dispersed by ingestion of seeds by animals are genetically more variable within the population.
86 4.5.4 Genetic distance and identity of populations of V. keniensis and V. Jischeri
The degree of similarity and dissimilarity between the different Vitexpopulation s were estimated by Nei's genetic statistics (1972), modified Rogers genetic distance (Wright, 1978) and Prevosti geneticdistanc e (1978). OnNei' s genetic statistics (1978) the similarities between the populations were high ranging from 0.980 to 0.989. The highest genetic similarity was recorded between Nyambene hills and Lake Victoria. Themea ngeneti cdistanc ebetwee nth e Vitexpopulation s onNei' s genetic statistics was 0.984. The genetic distance based on Nei's statistics was between 0.011 and 0.020. The highest level genetic distance wasobserve d between Mt. Elgonan d Lake Victoria (Table 4.6). The analyses by Modified Rogers genetic distance (Wright, 1978) and Prevosti genetic distance (Wright, 1978)exhibite d genetic distance patterns similar to those from Nei's genetic statistics (Table 4.7).
The pattern of variation in genetic identity and distance between the Vitex populations is random and does not follow the geographical distances, prevailing ecological conditions or the existing taxonomic delimitation. The highest divergence wasobserve d between Mt. Elgonan d LakeVictoria . This isi ncontras t toth e fact that the two populations are geographically closer to each other, occupy relatively similar ecological zones andhav ebee nconsidere d tob eth esam especie s(V. fischeri) differen t from the Nyambene hills and Mt. Kenya populations (V.keniensis) (Verdcourt, 1992; Beentje, 1994).
Table 4.6. Matrix of genetic similarity and distance coefficients. Below diagonal: Nei (1972) genetic identity (I); Above diagonal: Nei (1972) genetic distance (D) of V. keniensisand V. fischeri populations over 12 loci.
Vitex Population 1 2 3 4 1 Mt. Kenya ** #* 0.015 0.015 0.016 2 Nyambene hills 0.985 ** * * 0.019 0.011 3 Mt. Elgon 0.985 0.981 ** * * 0.020 4 Lake Victoria 0.984 0.989 0.980 ** * *
Table 4.7. Matrix of genetic similarity (I) and distance (D) coefficients: Below diagonal: Modified Rogersdistanc e (Wright, 1978):Abov ediagonal : Prevosti distance (Wright, 1978).
Population 1 2 3 4 1 Mt. Kenya ***** .027 .028 .029 2 Nyambene hills .023 ***** .032 .025 3 Mt. Elgon .023 .026 ***** .035 4 Lake Victoria .023 .020 .026 *****
87 Figure 4.4. UPGMA dendrogram of the four populations of V. keniensis and V. fischeri. Distance .02 .00 -H H + i Mt. Kenya H | i Mt. Elgon I | H| Nyambene hills Lake Victoria
The current taxonomic separation of the four populations of Vitex into V. keniensis (Mt. Kenya and Nyambene hills) and V. fischeri (Mt. Elgon and Lake Victoria) also does not appear to be reflected in the genetic distances or in the morphological traits (Chapter 2) of the populations. The distribution of genetic similarity and distance among the four Vitex populations did not follow any consistent pattern, an indication of their being conspecific.
In a review of electrophoretic analysis and plant systematics, Gottlieb (1977) concluded thatmos tconspecifi c populations havehig hmea ngeneti c identities on Nei's geneticstatistic s (1972),usuall y above0.90 , whilecongeneri cplan t specieshav elowe r genetic identities varying from 0.5 to0.6 . The mean genetic identity of the four Vitex populations was 0.984 which is well above congeneric level of divergence. It is well withinconspecifi c level. Themea ngeneti c distanceo f0.01 6als oseem s toindicat etha t the populations of the species have not yet diverged to species level.
The dendrograms of the genetic relationships between the Vitexpopulation s are illustrated in figures 4.4 and4.5 . Figure 4.4 isclustere d by theunweighte d pair group of arithmetic means (UPGMA) of Sneath and Sokal (1973), constructed using Prevosti genetic distance (Wright, 1978). The longest branch has a genetic distance of 0.035 and a cophenetic correlation of 0.0691. Figure 4.5 Wagner Tree (Farris, 1972) clustered by rooting the dendrogram at the midpoint of the longest path. The total length of the path is 0.058 and the cophenetic correlation is 1.00. It is also clustered from the genetic distance matrices (Table 4.7).
Both UPGMA and Wagner Tree dendrograms yielded branches of similar pattern. They grouped the four populations into two; Mt. Kenya and Mt. Elgon on one side, and Nyambene hills and Lake Victoria on the other (Figure 4.4 & 4.5). This also is in contrast to the prevalent taxonomic separation of the populations, geographical distancesan dth eprevailin gecologica l conditionsi nthei rhabitat . The Vitexpopulation s exhibited highmea n genetic identity and lowmea ngeneti c distancesbetwee n them, an indication that they are remain conspecific despite the varying geographical and ecological range of distribution. This is also a further evidence that most of the variation exist within the populations rather than between them.
88 Figure 4.5. Rooted Wagner tree dendrogram of the four V.keniensis an d V.fischeri populations. Distance from root .00 .01 .02 + + + + + + + + + + + + + i Mt. Kenya H | i Mt. Elgon I | | Nyambene hills Lake Victoria
.00 .01 .02
4.6 Conclusion
The high genetic diversity in Vitexpopulation s can be attributed to high level of outcrossing, pollen and seed dispersal within thepopulation s by beesan dmonkey san d birds respectively, and by the apparent neutrality of the loci to natural selection. Outcrossing species tend to have high genetic diversity evident from increased polymorphism, allelic diversity andheterozygosit y (Hooper &Haufler , 1997). Selfing species are more prone to lower levels of intra-population genetic diversity than outcrossing species, and in selfing species each population tends to be more differentiated from the others. According to Barrett & Shore (1989) and Crawford (1983) selfing plantsten dt ohav emor egeneti cvariatio nbetwee nthei rpopulation s than withinthe m because different alleles arefixe d during inbreeding. Ifthes e observations arecorrec t then, although Vitexha sa mixe dmatin gsystem , out-crossing predominates. Therefore explaining the high intra-population gene diversity (Hs) and the low inter- population gene diversity (DST) in Vitexpopulations . The high heterozygosity in the populations of Vitexcoul d also be attributed to the high polyploidy level (2n=96).
The low genetic variation between the disjunct populations of Vitexshow s lack of divergence a possible indication that no transformation of genetic constitution has occurred. Thedivergenc e ofpopulation smostl ydepend so nth eorigina lgenepool , time since separation, presence of natural barrier, ecological gradient (selection pressure) and physiological andphenotypi c plasticity ofth especies . According toth ehistor y and biogeography of the vegetation of East Africa the region was composed of a uniform plain during the Kainozoic Era (Haughton, 1963;Lin d & Morrison, 1974). That was changed by tectonic and volcanic activities that led to the formation of rift valleys and mountains during Miocene and Pleistocene periods (Lind & Morrison, 1974), thus separating the once continuous vegetation cover on the extensive plain. Therefore iti s possible that the geographically isolated Vitexpopulation s were once extensive and continuous, thus sharing a common ancestry. However, in spite of having been separated by the geological events, it seems they have not undergone sufficient drift for detectable morphological and genetic changes to warrant their taxonomic separation. The genetic relationship between the Vitex populations does not follow the current taxonomic separation and the level of genetic identity between them is well
89 above 0.90 (Table 4.6, Figures 4.4 & 4.5) implying that they are conspecific.
The study has shed some light on biogeographical history of the species by concluding that the Vitex populations are relicts of aonc e larger morewidesprea d one. The lack of unique alleles or combination of alleles across loci also supports a once larger, more widespread distribution. The ability of the species' populations to maintain close morphological and genetic similarity could be attributed to factors driving the evolutionary process such as the species gene-pool, time, selection pressure, gene-flow and biological adaptability which include polyploidy and phenotypic plasticity. These features can allow a species occupy different habitats without the need to undergo genetic or conspicuous morphological change. It is also possible that the variation in the selection pressure between the different ecological zones inhabited by the Vitex populations may not be as wide as it appears, therefore allowing or sustaining the homogeneity among them. However, little is known about the nature and magnitude of selective factors acting across the Vitexpopulations .
According to Allphin et al. (1998), high levels of heterozygosity tend to buffer genotypes against environmental challenges. The phenomenon can allow species to inhabit a wide range of ecological zone and slow down the pace of genetic change in species' populations. Other reports indicate that polyploids have a wide range of geographical distribution relativet odiploid s (Soltis& Soltis , 1991).Tha t could further explain the capacity of Vitex populations to occur in contrasting ecological zones. The long life span of the tree species (over 100 years) can also lead to time lag in transformation of demographic and genetic structure, slowing down the rate of population divergence. The high genetic identities and low genetic distances between the populations of Vitex populations therefore implies common ancestry and minimal or lack of divergence.
The high genetic variation within the Vitex populations, a possible sign of the absence of directional selection favouring particular genotypes, which can reduce heterozygosity. The species exhibited mixed mating system i.e. both out-crossing and selfing (Chapter 3), out-crossing appear to predominate thus maintaining the high heterozygosity in its populations. The high genetic variation within the populations seem to be maintained by efficient geneflow too within the populations through pollination and seed dispersal. It is therefore possible that the pollinators (bees) and seed dispersal agents (monkeys and hornbills) ensure extensive distribution of the genetic materials. The low inter-population variation between the Vitexpopulation s concurs with the observation by Hamrick & Godt (1990) that geographic range has little influence on inter-population differentiation of species. It appears that so long as the morphological, physiological and genetic characteristics of an organism do not impose limitations to its survival in an environment, evolution or speciation would be a luxury, unfortunately nature operates only onnecessities . That could explain thelac k of synchrony between the pattern of genetic structure, the ecological gradient and geographical distances of the Vitex populations.
90 5. General conclusions and recommendations
In spite of the ecological and geographical different inthei r habitats, the morpholo gical and genetic make-up of the Vitex populations appear similar. The studied traits of all the species' populations on the data from the available literature, herbarium specimens, morphology of freshly collected samples from the populations, cytology, mating systems and genetic structure revealed no reason for taxonomic separation of them into two species (V. keniensis and V. fischeri). Therefore V. fischeri and V. keniensisshoul d be considered as one species. The name V.fischeri ha s priority and the well-known V.keniensis fall s into synonymy. This supports the observation made by Dale & Greenway (1961) that V.keniensis an d V.fischeri ar e not distinguishable. The resemblance in the morphological characteristics of the two species by different authors presented in appendix 2.1 leads to a similar conclusion.
Thegeneti c identitiesbetwee nth e Vitexpopulation s (1= 0.984) werewel l withinth e range expected of conspecific populations (e.g. Crawford, 1983;Allphi n et al. 1998). Therefore any efforts for improvement breeding and conservation on programmes on the famous Meru oak, any of the four populations would adequately represent the species' genepool. However, the variation in timber properties between the Vitex populations needs further research, although it is probable that the timber traits are more under environmental influence. Theresult s of suchwor k would contribute to the improvement of the quality of the timber produced by the species.
The Vitex populations exhibit amixe d mating system and produces seeds by selfing as well as through out-crossing. This could be an adaptation against the possibility of missing apollinato r or amate . The seeds from self-pollination showed nomarke d loss in germination. This could be attributed to the high ploidy level and high heterozygo sity of the species. The two phenomena are capable of cushioning inbreeding depression. However, many generations of inbreeding can lead to vigour loss and reduced adaptability to the prevailing environmental conditions. More research is necessary to investigate the vigour of the selfed offspring throughout a complete life cycle. Sucha n investigation was notpossibl e at this stagebecaus e of the long life span of the tree species. The mixed mating system in the species allows both selfing and out-crossing. This may be considered advantageous in improvement breeding efforts, as it can allow exploitation of the species' heterozygosity and at thesam e time making it possible to benefit from the positive aspects of selfing such as the breeding of pure lines.
The tree species is pollinated mainly by bees (A. mellifera), therefore populations of these insects are vital for its survival. The current destruction of their habitats is likely to affect the pollinators adversely. Therefore, there is a need to preserve the species' natural habitatt omaintai npollinato r populations for enhanced geneflow inth e
91 Vitex populations. Otherwise reduced population of pollinators can lead to increased selfing and possibly inbreeding depression over generations thus threatening the species' survival. Besideshabita tpreservation , theexistin g bee-keeping cultureamon g the communities in the forest areas also need to be encouraged for the benefit of both the local population and the forest plant populations. The preservation of the habitat ofth etre e species isals ob e important protecting thepopulation s of the seeds dispersal agents i.e monkeys and hornbill birds. They play an important role in facilitating geneflow through seed dispersal. They alsopla y anessentia l role inpromotin g natural regeneration by pre-treatment of the seeds that pass through their digestive systems, enhancing germination.
The four Vitex populations had the same somatic chromosome number i.e. 2n=96. Thus they are polyploids. Their electrophoretic phenotypes and progeny segregation patterns indicates that they are tetraploids. Meanwhile, their progeny arrays exhibits multiple alleles of homozygotes, balanced and unbalanced heterozygotes. The independent segregation patterns are normally associated with autopolyploids (Lewis, 1980). Therefore, the populations of Vitex are autotetraploids.
Theleve l of total geneticdiversit y (HT=99%) estimated for Vitex washigh . Reports on the total genetic diversity of other polyploid tropical tree species indicate that Acacia karroo has 88% (Oballa, 1993) and Madura pomifera has 73% (Schnabel et al., 1991). From empirical evidence, it is possible to attribute the high genetic diversity in the Vitex populations to the presence of multiple alleles arising from the polyploidy. The genetic diversity may also maintained by the species' mixed mating systems and extensive geneflow through pollen and seed dispersal within the populations. The high morphological and genetic similarity between the populations couldb e attributed toth e Vitex populations having acommo norigin , uniform selection pressure, long life span of the tree species and other biological features such as phenotypic plasticity, heterozygosity and polyploidy. Protein polymorphism is often correlated to fitness (Mitton, 1994), therefore the high total diversity could be a reflection of the species' ecological adaptability. The high level of genetic diversity in the Vitexpopulation s reflect both outcrossing mode of reproduction and the potential longevity of the individual trees. The reasonably high levels of the existing genetic variation suggests that conservation efforts could still preserve most of the species diversity. However, it iswort h noting as acautio n that if the species isforce d to resort to inbreeding over generations, genetic erosion is likely to set in, leading reduced genetic diversity and loss of vigour.
Themaintenanc e of thegeneti cdiversit y isconsidere d crucial for long term survival andevolutionar y responseo fpopulation s tochange s inenvironmen t (Huenneke, 1991). Loss of genetic variation may reduce a population's ability to adapt to changing environmental conditions and lead to inbreeding depression. Despite the high genetic diversity inth e Vitex populations, their natural range isunde r increasing pressure from human activities and it isbecomin g impossible for itt ob emaintaine d indefinitely. The on-going selective harvesting in the species natural populations can also lead to
92 dysgenic effects, resulting loss of the best genotypes. Therefore, a concerted effort towards in-situan d ex-situ conservation isnecessar y for preserving the stability of the species' genetic structure.
The information on the genetic structure and mating systems can be applied in the improvement breeding and raising ofproductiv e commercial plantations of the species to reduce the over-exploitation of the natural population. The species' natural populations could be protected and managed as gene banks. The observed inter- population reproductive compatibility in Vitexi s advantageous because it can allow easy transfer of desirable inheritable traits from one population to another. However, the possibility of cross-pollination in the species implies that the designing of the seed orchards should ensure a separation distance from neighbouring unimproved individuals of the same species to avoid possible contamination through cross- pollination.
The variation between the Vitexpopulation s was low (GST= 3.5%) implying lack of population divergence. This is in contrast to the variation in the ecological and geographical distancesbetwee nth efou r disjucntpopulation s (Figure2.3) . Thehabitat s of twopopulation s (Mt. Kenya and Nyambenehills ) may bedescribe d asmontan e rain forest, while Mt. Elgon and Lake Victoria occur in the wet wooded savanna. The Wagner Tree dendrogram divided Vitexpopulation s into two main cluster groups, representing theleve lo f isozymedivergence . However,judgin g from thesmal lgeneti c distance between them, seeds collected from at least one of the populations would be enough to capture the majority of the genetic variation held within the species. Sampling intensity within the selected populations should be relatively high. This is because most of the species genetic diversity is retained within the populations. However, although there are no marked genetic differences between the populations, there is need to include the contrasting ecological zones in the conservation by including one from montane and the other from savanna forests as a precautionary measure, just in case some ecological-specific unique genetic traits were not detected by the isozyme technique.
Population genetic analyses of a threatened and highly exploited plant species such as Mem oak need a well-designed conservation programmes that would ensure preservation of maximum levels of natural genetic variation and local adaptation. Habitat fragmentation and loss of the species' natural populations is in the increase especially in the highly settled areas. This isboun d to expose the species to decline in its genetic diversity and adaptability to the dynamic environment. Genetic erosion would also impose a limitation to the potentials of the species' improvement through breeding. The debate on the optimum sampling intensity for conservation of genetic diversity is on-going. In the absence of resource constraints, the more the sample size the higher the chances of preserving the species' genetic diversity. However, a minimum of one hundred genotypes sampled across the range of the chosen Vitex populations wouldb eadequat efo r conserving itsgene-pool . Thisi sbecaus eth e species is an autotetraploid with a maximum of four alleles per locus, therefore according to
93 the formulae of Waples (1988) the expected number of genotypic categories is a hundred. It is worth stressing that if resources allow assembling a higher number of genotypes would increase the chances of capturing more of the species' gene pool.
The distance between the sampled individuals is also critical and the optimum isolation distance is dictated by the neighbourhood size which depends on the extent of pollen and seed dispersal. An isolation distance of 300 m, being the flight distance of the pollinating bees (Boshier et al., 1995), between sampled trees would be recommended for Vitex. The seed dispersal range by monkeys and hornbill birds in these environments is still unknown. These need to be established for the purpose of improving the sampling strategy for the species' genepool.
Since most of selection by users is based on adaptability to site and expected end uses such as timber, fruits and fuelwood, more research is necessary on the desirable traits and their heritability for improvement purposes. The observed variation inwoo d properties need to be investigated further to exploit its potential. This would promote the production and utilisation of the species both at subsistence and commercial level.
94 Appendix 2.1. Comparison of V. fischeri and V. keniensis made by different authors.
Table A.l. Comparison of V.fischeri an d V.keniensis by Dale & Greenway (1961). They concluded that collections away from Mt. Kenya are referable to as V.fischeri but it is doubtful if V. keniensisi s distinguishable from that species.
Characteristics V. fischeri V. keniensis
Height 30 ft 100 ft with 50 ft clear bole and 6 ft diameter.
Bark grey thin, rough and slightly fissured
Slash white blaze cream yellow
Young branches clothed with orange tawny — tomentose
Leaves 5-foliolate ...
Petiole 2.5 to 5.5 inches long ...
Leaflets entire, scabrous & puberulous above when young; scabrous & almost glabrous when mature, tomentose beneath, ovate-elliptic to elliptic or slightly obovate, 2.5 inches long, 1.25 to 2.5 inches broad; apex acuminate, base broadly cuneate to rounded, petiolule up to 0.5 inch long
Cymes axillary, densely flowered ... peduncles 3.5 inches long
Flowers pubescent, white mauve ...
Fruits oblong-globose, 0.4 inches long, black when ripe, cupped in enlarged membraneous calyx.
Habitat wet savanna; altitude 4000 to east of Mt. Kenya; altitude 6000 ft 5000 to 6000 ft
95 Table A.2. Comparison of V. fischeri and V. keniensis by Verdcourt (1992). He observed that V.keniensis isusuall y synonymised withV. fischeri bu t there isn odoub t that it is a distinct taxon worthy of specific rank.
Characteristics V. fischeri V. keniensis
Height Savannah shrub or small to Tree 12 - 30 m. fairly large tree (1.8-) 4.5 - 9 or sometimes to 1 5(- 18) m with rounded crown.
Diameter ... 1.8 - (2.3) m.
Clear bole ... 12 - 18 m.
Bark grey to dark brown shallowly very thin, rough and slightly serrate and fissured. fissured.
Slash white to light brown with creamy yellow turning dirty white sapwood green.
Stems & petiole young branches clothed with beneath with more shaggy velvety orange-brown hairs or indumentum than V. fischeri yellow tomentose
Leaves scented, 5-foliolate 5-foliolate
Leaflets oblong, ovate-elliptic to elliptic obovate, 5.5 - 17 cm. long, or slightly obovate elliptic, 5- 3.2 - 8.5 cm. wide, broadly 19 cm. long, 3-10 cm. wide, rounded to obtusely shortly to long-acuminate at acuminate at the apex, the apex, tip acute, cuneate to cuneate to rounded at the rounded at the base,entire base, coriaceous, usually scabrid-puberulous above but drying darker than V. fischeri, velvety woolly tomentose sparsely puberulous above, beneath with sparse to dense paler beneath and completely glands, venation evident, covered with soft ochraceous lateral veins rather numerous tomentum and glands. up to ± 15 veins.
Petioles 6.5-16.5 cm. long, petiolules 13.5 - 17 cm. long, petiolules 1-2 (3-8) cm. long. present or absent or interme diate 0.6-4 cm. long.
Cymes axillary, extensive, many ochraceous, tomentose, more flowered, up to 11 (-15 ) cm. lax than V. fischeri, axillary wide panicle up to 12 cm. long 24 cm. wide.
Peduncle 6-14 cm. long, pedicels 0-1 6.3 - 13 cm. long, secondary mm. long or that of central branches to 4.5 cm. long, flower of cyme up to 12 mm. pedicels 1-4 mm. long, All axes woolly tomentose densely tomentose.
Bracts 0.3-2.2 cm. long, dark, vel 0.5 - 1.0 cm. long, 1 - 4 mm. vety outside wide.
96 Calyx tinged red, 3-4 mm. long, densely ochraceous tomen- truncate or shallowly toothed, tose, more distinctly toothed teeth up to 1 mm. long than V. fischeri.
Tube 2 mm. long, lengthening to 5 - 6 mm. in fruit; teeth broadly triangular, 0.5 mm. long, lengthening to 3 mm.
Corolla white with blue or mauve white or white tinged blue upper lips and lower cream or mauve upper lips and with both blue; throat yellow; tube largest lobe mauve, tube ±3 mm. long, the lip 3.5 mm. curved, 5-6 mm. long, long and lower lobe 2.5 mm. ochraceous tomentose above long and wide. outside; limb 8-10 mm. wide, and largest lobe round or ovate, 3 - 5 mm. long, 3.5 mm. wide, entire, the others 2.5 - 3 mm. long 2 mm. wide, slightly emarginate, densely ochraceous tomento se outside.
Ovary — globose, with simple hairs and ovary
Style ... filiform, 6 mm. long
Stigma ... lobe-linear
Drupes Black or purple green to black Black with white spots, with green spots, oblong- obovoid, (1.1-) 2.2 - 2.4 cm. globose, 1-1.3 mm long sic, long, (0.8-) 1.3-1.5 cm. 0.8-1.0 cm. wide, edible. wide, shiny, glabrous, rounded at the apex, ± con tracted at the base.
Mesocarp calycine cup ± 1 cm. wide. fleshy, endocarp very woody, the putamen ±2 cm. long, 1.1 -1.2 cm. wide, 6 - 7 mm. diameter at the base, obtusely 4-angled.
Habitat Wooded grassland and thicket Evergreen forest, sometimes particularly on granite rocks, thicket particularly riverine; also rich bushland on termite 1290-2100 m. mounds and Bridelia, Maeso- psis, Albizia forest; 980-2080 m.
97 Table A.3. Comparison of V.fischeri an d V. keniensisb y H. Beentje (1994).
Characteristics V. fischeri V. keniensis
Height 3 - 15 m 12-35 m
Leaves 5-foliolate 5-foliolate
Leaflets elliptic or slightly obovate, elliptic, base often unequal, terminal leaflet 9-21 by 4 - 9 terminal, apex short cm slightly sand papery or acuminate, terminal leaflet 9- rarely glabrous above, densely 21 by 3.5-10 cm; slightly pubescent beneath, apex sand papery above, densely shortly acuminate. pubescent beneath.
Flowers white or pale mauve, with white or purplish, with largest lower lip darker mauve in lobe dark mauve, in axillary axillary dichasia 5 - 24 cm dichasia 12-18 cm long; long, flowers 6 - 8 mm long. flowers 7 - 8 mm long.
Fruits Purple to black, oblong- black, ellipsoid, 13-16 mm globose 8-12 mm long, long. edible.
Habitat wooded grassland rarely in moist evergreen forests. forest margins.
98 Appendix 2.2. Climatic conditions in the natural range of V. keniensis and V. fischeri populations. Sources: Forest station ledgers, Jaetzold & Schmidt (1982), Langdale-Brown et al.(1964) and Lind & Morrison (1974).
Mt. Kenya Region. Altitude: 1300 - 2000 m.
Month J F M A M J J A S 0 N D Annual
Temperature Mean 21 22 22 23 22 21 20 20 21 22 22 21 21.4 (°C) Max. 28 30 29 28 27 26 25 26 28 29 27 26 27.4 Min. 13 14 16 17 16 15 14 14 14 15 16 15 14.9
Mean rainfall (mm) 75 63 117 444 369 99 75 81 69 231 252 132 2007
Mean potential 174 171 189 153 138 120 114 126 159 180 144 138 1806 evaporation (mm)
Nyambene hills. Altitude: 1 300 - 2200 m.
Month J F M A M J J A S 0 N D Annual
Temperature Mean 16 16 17 16 16 14 13 13 14 16 16 15 15.2 (°C) Max. 22 23 22 21 20 19 17 17 20 21 20 21 20.3 Min. 9 10 11 12 11 10 9 9 9 11 11 10 10.2
Mean rainfall (mm) 45 60 93 315 294 63 57 54 51 207 201 81 1521
Mean potential 120 123 138 105 102 87 78 84 114 129 96 102 1278 evaporation (mm)
Mt. Elgon region. Altitude 1200 - 1500 m.
Month J F M A M J J A S 0 N D Annual
Temperature Mean 20 20 20 20 19 18 18 18 18 19 19 19 19.0 (°C) Max. 26 27 27 26 25 24 23 22 25 25 25 25 25.0 Min. 13 13 13 14 13 12 13 13 12 13 13 13 13.0
Mean rainfall (mm) 33 45 82 146 184 120 168 224 123 75 67 52 1419
Mean potential 165 154 164 135 115 105 102 108 123 136 129 152 1588 evaporation (mm)
Lake Victoria region. Altitude 1000 - 1400 m.
Month J F M A M J J A S 0 N D Annual
Temperature Mean 23 23 23 23 23 22 22 22 23 23 23 22 22.7 (°C) Max. 31 31 31 29 29 29 29 29 30 31 30 30 29.9 Min. 14 15 16 16 16 15 15 14 14 15 15 14 14.9
Mean rainfall (mm) 72 101 120 187 130 81 78 89 71 66 108 73 1176
Mean potential 180 160 180 150 140 130 140 150 160 170 160 170 1890 evaporation (mm)
99 Appendix 4.1. The enzymes screened on V. keniensisan d V. fischeri and their respective recipes. They are named according to IUB (1984).* indicates the best enzymes finally used in the analysis.
1. Acid Phosphate (ACP, E.C. 3.1.3.2) 0.1 M Acetate buffer pH 4.8 50 ml x-naphthylene acid phosphate 50m g Fast Garnet GBC 50m g
*2. Alcohol Dehydrogenase (ADH, E.C . 1.1.1.1) 0.1 M Tris-HCl pH 8.0 100m l 95% Ethanol 1.0 ml NAD 10m g MTT 10 mg PMS 2mg
*3. Diaphorase (DIA, E.C. 1.6.99.-) 0.05 M Tris-HCl pH 8.0 100m l 2,6 Dichlorophenol-indophenol 0.2 mg NADH 10.0 mg MTT 5.0m g
4. a-Esterase (a-EST, E.C. 3.1.1.-) 0.1 M Tris-HCl pH 7.25 100m l a-Napthyl acetate 100m g Fast blue RR salt 100m g
5. Glutamate dehydrogenase (GDH, E.C. 1.4.1.2) 0.1 M Tris-HCL pH 8.0 100m l L-Glutamic acid 1.0 g NAD 1.0 ml/10 mg MTT 1.0ml/1 0 mg PMS 0.5 ml/5 mg
6. Glucose-6-phosphate dehydrogenase (G-6-PD, E.C. 1.1.1.49) 0.1 M Tris-HCl pH 7.25 100m l D-Glucose-6-phosphate (disodium salt) 50 mg NADP 1.5 ml/15 mg MTT 1.5 ml/15 mg PMS 0.3 ml/3 mg
100 *7. Gluterate Oxaloacetate Transaminase (GOT, E.C. 2.6.1.1) 0.1 M Tris-HCl pH 8.35 100m l L-Aspartic acid 100m g a-Ketogluteric acid 33m g Pyridoxial-5-phosphate 4 mg PVP Soluble 50 mg Fast Blue BB Salt 100m g
8. Leucine-amino peptidase (LAP, E.C. 3.4.11.1) A 0.2 M Tris-HCl pH=7.5 50 ml Malic acid 200 mg Reduce pH to 6.0. Fast Garnet GBC 50 mg (Stir and filter) B Distilled water 750/x l Acetone 750 ^1 L-leucy1-b-naphth ylamide , HC1(endonuclease ) 15m g Add A to B pour over the gel and incubate at 25°C .
9. Malate dehydrogenase (MDH, E.C. 1.1.1.37) 0.1 M Tris-HCl pH 8.0 100m l 1.0 M DL-malic acid 3.0 ml NAD lOmg/l.Oml MTT lOmg/l.Oml PMS 3 mg/0.3 ml
*10. Malic Acid (ME, E.C. 1.1.1.40) 0.1 M Tris-HCl pH 8.0 100m l 1.0 M DL-Malic acid pH 7.0 3.0 ml NAD 10.0 mg MTT 10.0 mg PMS 3.0m g MgC12 100m g
*11. Manadione reductase (MR, E.C. 1.6.99.-) 0.1 Tris-HCl pH 7.25 100m l Manadione (sodium bisulphate) 20 mg NADH 10m g MTT 20 mg
101 *12. 6-PhosphogluconateDehydrogenas e (6-PGD, E.C. 1.1.1.44) 0.1 M Tris-HCl pH 7.5 100m l 1.0 M Magnesium chloride 1 ml NADP 30m g MTT 40 mg PMS 8 mg
*13. Phosphoglucoisomerase (PGI, E.C. 5.3.1.9) A 0.05 M Tris-HCl, pH 8.0 50 ml NAD 10m g Fructose-6-phosphate, Na salt 20 mg Glucose-6-phosphate dehydrogenase 20 units B MTT 20 mg PMS 8m g Add A to B, pour over gel and incubate.
14. Phosphoglucomutase (PGM, E.C. 5.4.2.2) A 0.1M Tris-HCl, pH 7.5 100m l 1M MgC12 1 ml Glucose-1-phospahte, sodium salt 150m g Glucose-6-phosGlucose-6-phosphate dehydrogenase 20 ix\ B MTT 20 mg NADP 20 mg PMS 8m g
*15. Shikimate Dehydrogenase (SDH, E.C. 1.1.1.25) 0.1 M Tris-HCl pH 8.0 100m l Shikimic acid 50 mg NADP 15m g MTT 15m g PMS 4 mg
102 Appendix 4.2. Genetic variability in each of the V. keniensis and V. fischeri populations at the different polymorphic loci derived from allelic frequencies.
Population: Mt . Kenya
Alleles Locus
ADH, ADH2 ADH3 DIA2 GOT2 GOT3 MR, ME, SDH2 6PGD PGI, PGI2 2 A .080 .105 .095 .095 .090 .075 .080 .110 .070 .075 .080 .085 B .085 .115 .090 .090 .080 .075 .090 .110 .100 .110 .075 .070 C .080 .080 .085 .100 .095 .100 .100 .085 .105 .100 .085 .080 D .085 .055 .070 .090 .085 .090 .115 .060 .125 .090 .070 .095 E .070 .045 .055 .020 .050 .060 .015 .030 .000 .025 .055 .050 F .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .025 .015
He .973 .969 .973 .969 .972 .972 .967 .969 .963 .968 .977 .975
Population: Nyambene hills
Alleles Locus
ADH, ADH2 ADH3 DIA2 GOT2 GOT3 MR, ME, SDH2 6PGD PGI, PGI2 2 A .970 .080 .085 .090 .080 .070 .080 .100 .080 .080 .090 .070 B .100 .085 .075 .090 .070 .100 .085 .110 .075 .100 .075 .085 C .075 .125 .095 .100 .105 .095 .100 .090 .115 .090 .080 .110 D .085 .050 .080 .085 .090 .080 .120 .080 .130 .095 .105 .065 E .070 .045 .070 .035 .055 .055 .015 .020 .000 .040 .045 .065 F .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .005 .005
He .972 .971 .972 .970 .971 .972 .967 .968 .963 .970 .972 .972
Population: Mt . Elgon
Alleles Locus
ADH, ADH2 ADH3 DIA2 GOT2 GOT3 MR, ME, SDH2 6PGD PGI, PGI2 2 A .080 .095 .095 .080 .095 .050 .095 .100 .085 .070 .085 .080 B .065 .090 .080 .105 .080 .100 .090 .125 .115 .110 .080 .070 C .075 .110 .090 .085 .100 .095 .095 .105 .105 .090 .075 .085 D .095 .065 .085 .075 .080 .080 .080 .055 .095 .105 .080 .085 E .085 .040 .050 .055 .045 .070 .045 .015 .000 .025 .050 .060 F .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .025 .030
He .972 .970 .972 .972 .971 .972 .970 .965 .964 .968 .976 .975
103 Population: Lake Victoria
Alleles Locus
ADH, ADH2 ADH3 DIA2 GOT2 GOT3 MR, ME, SDH2 6PGD PGI, PGI2 2 A .060 .090 .095 .105 .090 .080 .090 .090 .090 .070 .075 .075 B .080 .095 .065 .080 .055 .075 .075 .135 .085 .115 .085 .090 C .105 .095 .080 .105 .105 .105 .110 .095 .095 .090 .075 .105 D .085 .055 .105 .080 .095 .080 .100 .060 .130 .080 .110 .065 E .070 .065 .060 .030 .055 .055 .025 .020 .000 .040 .045 .050 F .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .010 .015
He .972 .971 .971 .969 .971 .972 .968 .965 .964 .971 .972 .973
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116 Acknowledgements
The author would like tothan k thefollowin g persons andinstitutions : Wageningen Agricultural University for having offered meth e sandwich fellowship. Prof. dr. ir. L.J.G. van der Maesen of the Laboratory of Plant Taxonomy and Prof. dr. R.F. Hoekstra ofth eDepartmen t ofGenetics , Wageningen Agricultural University forthei r valuable guidance in the formulation, implementation and finalisation of the PhD project. Dr. P. Oballa forhi ssupervisio n duringfield survey san dlaborator y analyses at Kenya Forestry Research Institute.
TheInternationa l Foundationo fScienc e(Stockholm-Sweden) , theAfrica n Academy of Sciences (Nairobi-Kenya) and German Agency for Technical Cooperation(GTZ ) supplied bothfinancial an dmateria l support. TheDirecto r ofKeny a Forestry Research Institute granted study leave andallowe d me tous eth einstitute' s resources duringth e field work. Iwoul d also like toexpres s mygratitud e toM sM . Buitelaar, Mr.X .va n der Burgt andMr .J . W.va nde rBur g forth esuppor t inBiosy s analysis, organisation of the text andscannin g respectively. Mytechnica l assistants David Meroka, Charles Oloo, Wakori, Joseph Munyao, Sylvester Odunga and Roseline Oluoch shared the burden of field andlaborator y work. Ia mgratefu l to field foresters including Esther Mbali (Migori), Mr. Dida forester Tarime (Tanzania), Mr. Gabriel Opoka of Uganda Forestry Seed Centre, foresters and forest guards of Nyambene, Meru, Chuka, Chogoria and Irangi for assisting inth efield work . Thedriver s Julius Njoroge, John Mwangi, Odunga and Oduor canb ecredite d withth e safe driving during theextensiv e field work to the various areas of the Meru oak in East Africa. I am grateful toth e Head (Dr. Khayota) and staff of the East African Herbarium, the curators of the following herbaria for loaning meVitex specimens:EA ,MHU , Kan d WAG. Mywif e Caroline and children Daisy, Dick and Roy have given me their moral support and their endurance over thelon g periods of my absence from home, for which Ia mver y thankful.
117 Curriculum vitae
Joseph OlooAhend a was born on23 rd December 1962 inSiaya , Kenya. Hejoine dth e Department of Forestry atMo i University inSeptembe r 1987fo r Bachelor of Science in Forestry and graduated in May 1990. He was employed by the Kenya Forestry Research Institute (KEFRI) inAugus t 1990a s anassistan t Research Officer based at the Forestry Seed Centre. In September 1992h ejoine d the Agricultural University, Wageningen for aMaste r of Science degree course inTropica l Forestry, having been awarded aGT Z (GermanAgenc yfo rTechnica lCooperation ) fellowship and graduated in January 1994. In April 1994 he was promoted to Research Officer in KEFRI. Besides conducting research work, he supervises undergraduate students from Moi University and organises courses in tropical forestry seed technology at KEFRI. He is a member of the Plant Genetic Resource Working Group inKenya . Heha scarrie d out a number of collaborative research with national and international organisations such as International Centre for Research in Agroforestry (Preparation of Primus africanamonograph) , International Plant Genetic Resource Institute (Conservationo f Veprisglandulosa), DANIDA Forestry Seed Centre (Recalcitrant and intermediate tropical forestry seed research), Regional Soil Conservation Unit (Designing and establishment of community tree seed stands), Regional Land Management (Training of agricultural and forestry extensionists), National Museums of Kenya (Population genetics andreproductiv e biology of Veprisglandulosa) andApplie d Research Uniti n Laikipia, Kenya (production ofcommunity - based forestry training materials). He has co-authored theTre e SeedHandboo kan dUsefu l IndigenousTree so fBungom a District (Kenya). In October 1995 he was offered a sandwich PhD fellowship by the Agricultural University, Wageningen under the supervision of Prof. Dr. L.J.G. van der Maesen andProf . R.F.Hoekstra . Hei scurrentl y aResearc h Officer atKeny ath e Forestry Research Institute near Nairobi in forestry genetics andsee d research.
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